Dual and triple hapten conjugates, compositions, methods of making, and methods of treatment therewith

Abstract

A conjugate having the formula TL-L-Hn, where TL is a targeting ligand for a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell, or a fibroblast cell, L is a linker, H is a hapten, and n is an integer equal to or greater than 2. Compositions comprising the conjugate and methods of use are also provided.

Description

[Technical field]

[0001] Priority This application is related to and claims the benefit of priority to (1) U.S. Provisional Application No. 63 / 332,521, filed April 19, 2022; (2) U.S. Provisional Application No. 63 / 392,744, filed July 27, 2022; and (3) U.S. Provisional Application No. 63 / 429,030, filed November 30, 2022. The contents of the foregoing applications are incorporated herein by reference to the present disclosure in their entireties.

[0002] Technical Field The present disclosure includes conjugates comprising two or more haptens linked to targeting ligands for target proteins on viruses or cells, as well as compositions (e.g., pharmaceutical compositions) comprising such conjugates, and methods for producing such conjugates. In addition, the present disclosure further includes methods for treating viral infections, fibrosis, and cancer. [Background technology]

[0003] This section introduces aspects that may be helpful to facilitate a further understanding of the present disclosure. Accordingly, these statements are to be read in this light, and are not to be understood as admissions about prior art or not.

[0004] Known Fc-mediated (fragment crystallizable domain-mediated) antibody effector functions are antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). In addition, antibodies have been found to mediate inflammation and immune regulation by inducing cell differentiation and activation. These mechanisms may protect viral replication or enhance the elimination of infected cells through antibody-mediated effector functions on virus-infected cells, such as the excretion of viral glycoproteins, internalization of viral glycoproteins, antibody cooperativity, and antibody glycosylation. The families of receptors that recognize the Fc domain of IgG (immunoglobin G) molecules are known as the FcRn family and the FcγR family. Several studies have demonstrated a correlation between the affinity and selectivity of Fc:FcγR and the cytotoxic function of immune effector cells involving immune complexes (ICs).

[0005] Human gamma receptors (FcγR) include FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIc (CD32c), FcγRIIIa (CD16a), and FcγRIIIb (CD16b), which are expressed at different levels on the surface of various immune cells. FcγRIIIa is an important surface receptor in terms of contributing to ADCC activity and is found on the surface of natural killer (NK) cells, macrophages, monocytes, mast cells, eosinophils, and dendritic cells. However, it is the only FcγR expressed by NK cells.

[0006] Humans express two FcγRIIIa allotypes that differ by a single amino acid at position 158; the residue can be either valine (V) or phenylalanine (F), with the V at 158 ​​isoform having high affinity for the Fc domain of IgG1 and the F at 158 ​​isoform having low affinity. Engagement of high affinity FcγRIIIa-V158 by immune complexes (IC) results in stronger in vitro cytotoxic potency compared to FcγRIIIa-F158. Fc-engineered antibodies with improved affinity for FcγRIIIa can prime and activate NK cells more efficiently, thus demonstrating enhanced therapeutic efficacy compared to native Fc. ADCP activity is known to be driven by FcγRIIa intracellular signaling, which has been shown by glycoengineered antibodies to show enhanced affinity for FcγRIIa and thus increased ADCP activity. Mainly, Fc variants that show high affinity for FcγRIIa-R131 isoform and high selectivity for FcγRIIa over FcγRIIb have been found to mediate improved ADCP activity. The phagocytic mechanism can involve either complement receptor. Infected cells can be eliminated by CDC as well as ADCC and / or ADCP mediated by FcγR-bearing effector cells.

[0007] Nearly all therapeutic antibodies approved by the FDA are full-sized IgG1 antibodies with a size of approximately 150 kDa. Dimitrov, Engineered CH2 domains (nanoantibodies), mAbs 1(1): 26-28 (2009). The same is true for the majority of antibodies in clinical trials. The exceptions are antigen-binding fragments (Fabs).

[0008] Full-sized antibodies present fundamental problems for therapy. One problem is poor tissue penetration, such as poor solid tumor penetration. Dimitrov (2009), supra. Another problem is weak or absent binding to regions on the surface of some molecules, such as the envelope glycoproteins of the human immunodeficiency virus (HIV). Ibid.

[0009] In view of the foregoing, what is needed are materials and methods for bringing antibodies (Abs) (whether naturally occurring, exogenously administered autologous Abs, or exogenously administered IgG Abs) into close proximity with viruses, virus-infected cells, cancer cells, immune cells, and / or fibroblasts, e.g., to improve Ab function. This and other objects and advantages, as well as features of the invention, will become apparent from the detailed description provided herein. Summary of the Invention

[0010] In one embodiment of the present disclosure, n wherein TL is a targeting ligand for a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell, or a fibroblast cell; L is a linker; H is a hapten; and n is an integer of 2 or 3, and pharma- ceutically acceptable salts thereof.

[0011] In a further aspect of the disclosure, the compound of formula IA:

[0012] [ka] and pharma- ceutically acceptable salts thereof, During the ceremony, TLs are targeting ligands for target proteins on the surface of viruses, virus-infected cells, cancer cells, immune cells, or fibroblasts, and L a , L b , and L c is a linker, C is a carbon atom, R4 is selected from hydrogen, a C1-C5 alkyl group, a C1-C5 alkenyl group, or a C1-C5 alkynyl group; H1 and H2 are haptens; Conjugates, and pharma- ceutically acceptable salts thereof, are provided.

[0013] In a further aspect of the disclosure, a compound of formula IB:

[0014] [ka] and pharma- ceutically acceptable salts thereof, wherein TL is a targeting ligand for a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell, or a fibroblast cell; and L a , L b , L c , and L d is a linker, C is a carbon atom, and H1, H2, and H3 are haptens, and pharma- ceutically acceptable salts thereof are provided.

[0015] In a further aspect of the disclosure, a compound of formula II

[0016] [ka] or a pharma- ceutically acceptable salt thereof, wherein L1, L2, and L3 are each independently a linker.

[0017] In certain embodiments, the conjugate has the formula: TL-LH n or a pharma- ceutically acceptable salt thereof, in which T is a targeting ligand for a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell, or a fibroblast cell, L is a linker, H is a hapten, and n is an integer from 2 to 3, and optionally at least two of H are each capable of binding to a different antibody when contacted with the different antibody.

[0018] At least two of H can each be bound by an antibody. Each H can be bound by a different antibody. In certain embodiments, at least two of H can each be bound by a different antibody when contacted with an antibody in vivo. In certain embodiments, at least two of H can each be bound by a different antibody when contacted with an antibody in vitro.

[0019] Each H may be independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof. At least one H may be an influenza virus antigen selected from hemagglutinin and neuraminidase. At least one H may be a hepatitis antigen selected from L-HBsAg, S-HBsAg, M-HBsAg, and preS. At least one H may be gp120 or gp160. At least one H may be a glycoprotein.

[0020] n may be 2. n may be 3.

[0021] Each H may be independently selected from a rhamnose fragment, an α-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin, or from an antigen of a virus selected from diphtheria, varicella zoster virus, human papilloma virus, influenza virus, SARS-COV-2, yellow fever, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis G, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox, monkeypox, poliovirus, dengue virus, and measles virus.

[0022] In certain embodiments, n is 2, the first H is a DNP fragment, and the second H is a rhamnose fragment.

[0023] The target protein can be a viral envelope protein or a viral envelope protein on the surface of a virally infected cell. The target protein can be influenza neuraminidase or influenza hemagglutinin. The target protein can be respiratory syncytial virus fusion protein F. The target protein can be a coronavirus spike protein. The target protein can be hepatitis B virus surface antigen or HBV core antigen. The target protein can be a cell surface receptor on a cancer cell. The target protein can be a folate receptor. The target protein can be folate receptor alpha or folate receptor beta. The target protein can be prostate specific membrane antigen.

[0024] The conjugate of any one of claims 1-3, 5, 6, or 9-12, wherein the target protein is carbonic anhydrase 9. The target protein can be a luteinizing hormone releasing hormone receptor. The target protein can be a neurokinin 1 receptor. The target protein can be a cell surface receptor on tumor associated macrophages. The target protein can be a cell surface receptor on myeloid derived suppressor cells. The target protein can be a cell surface receptor on cancer associated fibroblasts. The target protein can be a fibroblast activation protein. The targeting ligand can be a neuraminidase inhibitor. The targeting ligand can be an oseltamivir fragment, a zanamivir fragment, a peramivir fragment, or a laninamivir fragment. The targeting ligand can be a zanamivir fragment. The targeting ligand can be a folic acid fragment or an analog thereof. The targeting ligand can be 5-methyltetrahydrofolic acid.

[0025] L is (-CH2CH2-O-) n (wherein n is an integer from 1 to 32, inclusive), a peptide, a peptidoglycan, or a combination of two or more of the foregoing. L can be a branched linker, where at least two of the haptens are attached to different branches of the linker, and the different branches optionally extend from different atoms of the linker.

[0026] The targeting ligand may be a folate fragment or a derivative thereof, at least the first H may comprise a rhamnose fragment, and at least the second H may comprise a dinitrophenyl fragment.

[0027] The conjugates of the invention can be formulated as prodrugs.

[0028] In certain embodiments, the conjugate has the formula

[0029] [ka] or a pharma- ceutically acceptable salt thereof, in which TL is a targeting ligand for a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell, or a fibroblast cell; and L a , L b , and L c are each a linker which may be the same or different, C is a carbon atom, R4 is selected from hydrogen, a C1-C5 alkyl group, a C1-C5 alkenyl group, or a C1-C5 alkynyl group, H1 and H2 are each a hapten, and optionally H1 and H2 can each bind to a different antibody.

[0030] In certain embodiments, the conjugate has the formula

[0031] [ka] or a pharma- ceutically acceptable salt thereof, in which TL is a targeting ligand for a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell, or a fibroblast cell; and L a , L b , L c , and L dare each linkers which may be the same or different, C is a carbon atom, H1, H2, and H3 are each haptens, and optionally each H1, H2, and H3 can each bind to a different antibody.

[0032] H1 and H2 can each be bound by an antibody. H1, H2, and H3 can each be bound by an antibody. H1 and H2 can each be independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof. H1, H2, and H3 can each be independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof. H1 or H2 may each independently be selected from a rhamnose fragment, an alpha-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin, or from a viral antigen selected from diphtheria, varicella zoster virus, human papilloma virus, influenza virus, SARS-COV-2, yellow fever, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis G, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox, monkeypox, poliovirus, dengue virus, and measles virus. H1, H2, or H3 may each independently be selected from a rhamnose fragment, an alpha-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin, or from an antigen of a virus selected from diphtheria, varicella-zoster virus, human papilloma virus, influenza virus, SARS-COV-2, yellow fever, respiratory syncytial virus, herpes simplex virus, chickenpox virus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis G, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox, monkeypox, poliovirus, dengue virus, and measles virus. In certain embodiments, H1 is a DNP fragment and H2 is a rhamnose fragment.

[0033] The at least one hapten can be an influenza virus antigen selected from hemagglutinin and neuraminidase. The at least one hapten can be a hepatitis antigen selected from L-HBsAg, S-HBsAg, M-HBsAg, and preS. The at least one hapten can be gp120 or gp160. The at least one hapten can be a glycoprotein.

[0034] The target protein can be a viral envelope protein or a viral envelope protein on the surface of a virus-infected cell. The target protein can be influenza neuraminidase or influenza hemagglutinin. The target protein can be respiratory syncytial virus fusion protein F. The target protein can be coronavirus spike protein. The target protein can be hepatitis B virus surface antigen or HBV core antigen. The target protein can be a cell surface receptor on a cancer cell. The target protein can be a folate receptor. The target protein can be folate receptor alpha or folate receptor beta. The target protein can be prostate-specific membrane antigen. The target protein can be carbonic anhydrase 9. The target protein can be luteinizing hormone releasing hormone receptor. The target protein can be neurokinin 1 receptor. The target protein can be a cell surface receptor on tumor-associated macrophages. The target protein can be a cell surface receptor on myeloid-derived suppressor cells. The target protein can be a cell surface receptor on cancer-associated fibroblasts. The target protein can be a fibroblast activation protein. The targeting ligand may be a neuraminidase inhibitor. The targeting ligand may be an oseltamivir fragment, a zanamivir fragment, a peramivir fragment, or a laninamivir fragment. The targeting ligand may be a zanamivir fragment. The targeting ligand may be a folic acid fragment or an analog thereof. The targeting ligand may be 5-methyltetrahydrofolic acid.

[0035] In certain embodiments of this conjugate, L a , Lb , L c , and L d At least one of each independently represents (-CH2CH2-O-) n (wherein n is an integer from 1 to 32, inclusive), an alkyl group, a peptide, a peptidoglycan, or a combination of two or more of the foregoing. a , L b , and L c At least one of each independently represents (-CH2CH2-O-) n where n is an integer from 1 to 32, inclusive, an alkyl group, a peptide, a peptidoglycan, or a combination of two or more of the foregoing. n can be an integer from 1 to 16, inclusive.

[0036] L a , L b , and L c , and L d At least one of the L may comprise a peptide fragment or a peptidoglycan fragment. a , L b , and L c At least one of the L may comprise a peptide fragment or a peptidoglycan fragment. a , L b , and L c , L d are respectively independent of each other, C2 to C 18 It may contain an alkyl group. a , L b , and L c are respectively independent of each other, C2 to C 18 It may contain alkyl groups.

[0037] In certain embodiments, the conjugate has the formula:

[0038] [ka] or a pharma- ceutically acceptable salt thereof.

[0039] In certain embodiments, the conjugate has the formula:

[0040] [ka] or a pharma- ceutically acceptable salt thereof.

[0041] One or more of the -OH groups of the conjugate may be independently replaced with a thiol, phosphate, or phosphanate ester. One or more of the -OH groups may be replaced with -OC(=O)R, where R is an alkyl group. One or more of the -OH groups may be replaced with -OC(=O)R, where R is a C1-C6 alkyl group. An amine (-NH2) group may be replaced with -OC(=O)R2, where R2 may be an alkyl group. An amine (-NH2) group may be replaced with -OC(=O)R2, where R2 may be a C1-C6 alkyl group. A carboxyl (-COOH) group may be replaced with -OC(=O)R3, where R3 is an alkyl group. A carboxyl (-COOH) group may be replaced with -OC(=O)R3, where R3 is a C1-C6 alkyl group.

[0042] In certain embodiments, the conjugate has the formula:

[0043] [ka] or a pharma- ceutically acceptable salt thereof, wherein L1, L2, and L3 are linkers. One or more of L1, L2, and L3 are (-CH2CH2-O-) n (wherein n can be an integer from 1 to 16, inclusive). L1, L2, and L3 can each independently be C2 to C 18 It may include an alkyl group, a peptide fragment, or a peptidoglycan fragment.

[0044] In certain embodiments, the conjugate has the formula:

[0045] [ka] or a pharma- ceutically acceptable salt thereof.

[0046] The conjugate may be further conjugated in vivo to one or more antibodies.

[0047] Pharmaceutical compositions are also provided, which may include a conjugate (e.g., any of the conjugates of the invention) and a pharma- ceutically acceptable excipient.

[0048] Also provided is a method for treating a viral infection in a subject. The method for treating a viral infection in a subject may comprise administering an effective amount of the conjugate of the present invention or the pharmaceutical composition of the present invention to the subject. The method may further comprise administering an autoantibody or an allogeneic immunoglobulin G (IgG) antibody to the subject. The viral infection may be influenza. The conjugate or pharmaceutical composition may be administered orally. The conjugate or pharmaceutical composition may be administered once a day. The conjugate or said pharmaceutical composition may be administered multiple times a day. The conjugate or pharmaceutical composition may be administered twice a day.

[0049] A method for treating cancer in a subject is provided. The method for treating cancer in a subject may comprise administering an effective amount of the conjugate of the present invention or the composition (e.g., pharmaceutical composition) of the present invention to the subject. The method may further comprise administering an autoantibody or an allogeneic IgG antibody to the subject. The cancer may be a hot cancer. The cancer may be renal cancer, lung cancer, or colorectal cancer.

[0050] The conjugate or pharmaceutical composition may be administered orally or intravenously. The conjugate or pharmaceutical composition may be administered once daily.

[0051] The methods of treating cancer may further include administering to the subject a second therapeutic agent, wherein the second therapeutic agent comprises a chemotherapeutic agent, sunitinib, a PD-1 inhibitor, or a PDL-1 inhibitor.

[0052] Also provided is a method for activating the immune response of a subject, and in certain embodiments, such a method comprises administering to the subject an effective amount of the conjugate of the present invention or the pharmaceutical composition of the present invention.The immune response can be a natural immune response.The immune response can be activated in a target area of ​​the subject (e.g., tumor microenvironment, or the location of a viral replication site).

[0053] The method of activating an immune response may further comprise administering to a subject an autoantibody or an allogeneic IgG antibody. Administration of an effective amount of the conjugate or pharmaceutical composition may induce reprogramming of M2-type macrophages to M1-type macrophages in the target area.

[0054] Embodiments of the present disclosure, as well as other features, advantages, and aspects contained herein, and the basis for achieving the same, will become apparent in view of the following detailed description of various exemplary embodiments of the present disclosure, which will be better understood when taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0055] [Figure 1-1] FIG. 1A is a schematic illustration of antibody recruitment. [Figure 1-2] FIG. 1B is a schematic illustration of antibody recruitment using an embodiment of a conjugate of the invention, 10, which comprises a targeting ligand (TL) and two haptens (H each), where the targeting ligand is specific for neuraminidase (i.e., a receptor) of a virus or a virus-infected cell, and hapten H of conjugate 10 binds to human endogenous anti-DNP and anti-Rha antibodies (Abs 12). [Figure 2-1]Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectroscopy data for compound 7 (Figure 2A) are shown. [Figure 2-2] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectroscopy data for compound 7 (Figure 2A) are shown. [Figure 2-3] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectroscopy data for compound 8 (Figure 2B) are shown. [Figure 2-4] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectroscopy data for compound 8 (Figure 2B) are shown. [Figure 2-5] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectrometry data for compound 9 (Figure 2C) are shown. [Figure 2-6] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectrometry data for compound 9 (Figure 2C) are shown. [Figure 2-7] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectrometry data for compound 12 (Figure 2D) are shown. [Figure 2-8] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectrometry data for compound 12 (Figure 2D) are shown. [Figure 2-9] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectrometry data for compound 18 (Figure 2E) are shown. [Figure 2-10] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectrometry data for compound 18 (Figure 2E) are shown. [Figure 2-11] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectroscopy data for compound 22 (Figure 2F) are shown. [Figure 2-12] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectroscopy data for compound 22 (Figure 2F) are shown. [Figure 2-13]Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectrometry data for compound 23 (Figure 2G) are shown. [Figure 2-14] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectrometry data for compound 23 (Figure 2G) are shown. [Figure 2-15] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectroscopy data for compound 24 (Figure 2H) are shown. [Figure 2-16] Liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and ultraviolet spectroscopy data for compound 24 (Figure 2H) are shown. [Diagram 3] Graph of days post-infection vs. % survival for mice (n=5 / group) infected with 100 LD50 of Influenza A H1N1 / PR8, clearance of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg (GAMUNEX®-C)) at 24 hours post-infection (hpi), and administration of the conjugate at 48 hpi. [Figure 4] FIG. 5 is a graph of days post-infection versus body weight (%) for mice treated as described in FIG. [Diagram 5] Graph of days post-infection versus % survival for mice (n=5 / group) infected with 100 LD50 of Influenza A H1N1 / PR8, depletion of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg (GAMUNEX®-C)) at 24 hpi, and administration of the conjugate at 48 hpi. [Figure 6] FIG. 6 is a graph of days post-infection versus body weight (%) for mice treated as described in FIG. 5. [Figure 7] Graph of days post-infection versus % survival for mice (n=5 / group) infected with 100 LD50 of Influenza A H1N1 / PR8, depletion of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg (GAMUNEX®-C)) at 24 hpi, and administration of the conjugate at 96 hpi. [Figure 8]FIG. 8 is a graph of days post-infection versus body weight (%) for mice treated as described in FIG. [Figure 9] Graph of days post-infection versus % survival for mice (n=5 / group) infected with 100 LD50 of Influenza A H1N1 / PR8, depletion of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg (GAMUNEX®-C)) at 24 hpi, and administration of the conjugate at 48 hpi. [Figure 10] FIG. 10 is a graph of days post-infection versus body weight (%) for mice treated as described in FIG. [Figure 11] Graph of days post-infection versus % survival for mice (n=5 / group) infected with 100 LD50 of Influenza A H1N1 / PR8, depletion of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg (GAMUNEX®-C)) at 24 hpi, and administration of the conjugate at 96 hpi. [Figure 12] 12 is a graph of days post-infection versus body weight (%) for mice treated as described in FIG. [Figure 13-1] FIG. 13A shows the viral titers (fold change) for the conjugate and commercial drug 48 hpi. [Figure 13-2] FIG. 13B shows the viral titers (fold change) for the conjugate and commercial drug 96 hpi. [Figure 14-1] FIG. 14A shows the viral titers (fold change) for the conjugate and commercial drug 48 hpi. [Figure 14-2] FIG. 14B shows the viral titers (fold change) for the conjugate and commercial drug 96 hpi. [Figure 15-1] FIG. 15A is a graph of days post-infection versus % survival. [Figure 15-2] FIG. 15B is a graph of days post-infection versus body weight (%). [Figure 16]1 is a bar graph of route of administration (SC=subcutaneous; OG=oral gavage; IV=intravenous; IN=nasal; PBS=phosphate buffered saline) versus viral titer (PFU / ml per ng of ribonucleic acid (RNA)). [Figure 17] 1 is a bar graph of virus titer (PFU / ml per ng of RNA) versus OG and PBS. [Figure 18] FIG. 1 is a graph of time (h=hours) versus concentration (ng / mL) for a single intravenous (IV) dose of 1.5 μmol / kg relevant to a pharmacokinetic study of administration of compound 24 (Zan-DNP-rhamnose) (n=2 mice / time point). [Figure 19] FIG. 1 is a graph of time (h=hours) versus concentration (ng / mL) for a single oral dose of 4.5 μmol / kg relevant to a pharmacokinetic study of administration of compound 24 (Zan-DNP-rhamnose) (n=2 mice / time point). [Figure 20] Figure 20A is a graph of days post-infection vs. % survival associated with a dose escalation study, where mice were infected with 100xLD50 influenza A / H1N1 / PR8 / 1934, hIVIg was used as a source of anti-heptane antibodies (injected at a dose of 8g / kg 24 hours prior to drug administration), and a dose of compound 24 (Zan-DNP-rhamnose) was administered 48 hpi with a single dose of 1.5μmol / kg zan-DNP-rhamnose (IV) or two doses of 4.5umol / kg zan-DNP-rhamnose (oral). Figure 20B is a graph of days post-infection vs. % body weight associated with the dose escalation study of Figure 20A, where the legend of Figure 20A also applies to Figure 20B. [Figure 21] FIG. 1 is a graph of days post-infection versus viral titer (PFU / ml per ng of RNA) for PBS, and IV administration of zanamivir-DNP-rhamnose (compound 24) and OG. [Figure 22] Graph of days post-infection versus % survival. [Diagram 23] Graph of days post-infection versus body weight (%). [Figure 24]Graph showing lung titers in Balb / c mice 24 hours after treatment, 48 hours after infection (day 0) with 10LD50 A / H1N1 / PR8 / 1934 virus, comparing treatment with Compound 24 to PBS, Tamiflu®, Xofluza®, and a cohort of uninfected mice (no infection). [Diagram 25] FIG. 1 is a graph showing survival of Balb / c mice after infection with 10 LD50 A / H1N1 / PR8 / 1934 virus (day 0) and administration of Compound 24 (C.24) by various routes (i.e., IN, IV, and OG) on day 4 post-infection compared to PBS alone. [Figure 26] FIG. 26 is a graph showing % infection versus body weight based on the dosing data in FIG. 25. [Figure 27-1] FIG. 27A shows graphs of compound 24 concentration (nM) versus antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) killing rates on N1-transfected (NA-HEK) and wild-type (WT) HEK293 cells in vitro. [Figure 27-2] FIG. 27B shows graphs of compound 24 concentration (nM) versus antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) killing rates on N1-transfected (NA-HEK) and wild-type (WT) HEK293 cells in vitro. [Figure 28-1] FIG. 28A shows graphs of days post-infection vs. survival (%) and days post-infection vs. body weight (%) from a dose-escalating study of Compound 24 administered IV in Balb / c mice. [Figure 28-2] FIG. 28B shows graphs of days post-infection vs. survival (%) and days post-infection vs. body weight (%) from a dose-escalating study of Compound 24 administered IV in Balb / c mice. [Figure 29-1] FIG. 29A shows graphs of days post-infection vs. survival rate (%) and days post-infection vs. body weight (%) from a study involving administration of a conjugate of the invention compared to monohapten (treatment 96 hpi) in Balb / c mice. [Figure 29-2] FIG. 29B shows graphs of days post-infection vs. survival rate (%) and days post-infection vs. body weight (%) from a study involving administration of a conjugate of the invention compared to monohapten (treatment 96 hpi) in Balb / c mice. [Figure 30-1] FIG. 30A shows graphs of days post infection vs. survival (%) and days post infection vs. body weight (%) from a study involving administration of the conjugate to Balb / c mice infected with various strains of flu, where (A) is the influenza A / California / 07 / 2009(H1N1)pdm09+Compound 24 treatment group; (B) is the influenza A / Wisconsin / 67 / 2005(H3N2)+Compound 24 treatment group; (C) is the influenza B / Florida / 04 / 2006+Compound 24 treatment group; (D) is the influenza A / Califonria / 07 / 2009(H1N1)pdm09+PBS control group; (E) is the influenza A / Wisconsin / 67 / 2005(H3N2)+PBS control group; and (F) is the influenza B / Florida / 04 / 2006+PBS control group. [Figure 30-2] FIG. 30B shows graphs of days post infection vs. survival (%) and days post infection vs. body weight (%) from a study involving administration of the conjugate to Balb / c mice infected with various strains of flu, where (A) is the influenza A / California / 07 / 2009(H1N1)pdm09+Compound 24 treatment group; (B) is the influenza A / Wisconsin / 67 / 2005(H3N2)+Compound 24 treatment group; (C) is the influenza B / Florida / 04 / 2006+Compound 24 treatment group; (D) is the influenza A / Califonria / 07 / 2009(H1N1)pdm09+PBS control group; (E) is the influenza A / Wisconsin / 67 / 2005(H3N2)+PBS control group; and (F) is the influenza B / Florida / 04 / 2006+PBS control group. [Diagram 31] Graphs of cytokine and chemokine levels measured in the lungs of treated or control mice are shown. [Figure 32-1] 1 shows graphs of serum cytokine and chemokine levels measured in treatment and control groups to monitor the presence or absence of systemic inflammation in test subjects. [Figure 32-2] 1 shows graphs of serum cytokine and chemokine levels measured in treatment and control groups to monitor the presence or absence of systemic inflammation in test subjects. [Diagram 33] 1 shows qualitative histological analysis images of lung tissue biopsies taken from mice infected with influenza A / H1N1 / PR8 / 1934 and treated with 1) PBS, 2) Compound 24 IV, or 3) Compound 24 OG. [Figure 34-1] 1 shows histological images of lung or kidney tissues of virus-infected mice with or without treatment with compound 24 (oral or intravenous). [Figure 34-2] 1 shows histological images of lung or kidney tissues of virus-infected mice with or without treatment with compound 24 (oral or intravenous). [Figure 35-1] 1 shows a synthesis scheme for compound 150, a folic acid dual hapten conjugate. [Figure 35-2] 1 shows a synthesis scheme for compound 150, a folic acid dual hapten conjugate. [Diagram 36] LC-MS, mass spectrometry, and UV spectroscopy data for compound 3' are shown. [Figure 37-1] LC-MS, mass spectrometry, and UV spectroscopy data for compound 5' are shown. [Figure 37-2] LC-MS, mass spectrometry, and UV spectroscopy data for compound 5' are shown. [Figure 38-1] LC-MS, mass spectrometry, and UV spectroscopy data for compound 10' are shown. [Figure 38-2] LC-MS, mass spectrometry, and UV spectroscopy data for compound 10' are shown. [Figure 39-1] LC-MS, mass spectrometry, and UV spectroscopy data for compound 11' are shown. [Figure 39-2]LC-MS, mass spectrometry, and UV spectroscopy data for compound 11' are shown. [Figure 40-1] LC-MS, mass spectrometry, and UV spectroscopy data for compound 13' are shown. [Figure 40-2] LC-MS, mass spectrometry, and UV spectroscopy data for compound 13' are shown. [Figure 41-1] LC-MS, mass spectrometry, and UV spectroscopy data for compound 14' are shown. [Figure 41-2] LC-MS, mass spectrometry, and UV spectroscopy data for compound 14' are shown. [Figure 42-1] LC-MS, mass spectrometry, and UV spectroscopy data for compound 18' are shown. [Figure 42-2] LC-MS, mass spectrometry, and UV spectroscopy data for compound 18' are shown. [Figure 43-1] LC-MS, mass spectrometry, and UV spectroscopy data for compound 150 are shown. [Figure 43-2] LC-MS, mass spectrometry, and UV spectroscopy data for compound 150 are shown. [Diagram 44] 1 shows data relating to a CDC assay shown as a graph of folate dual hapten (compound 150 = FDH) concentration (nM) versus CDC killing percentage, showing compound 150 administered to mouse lung cancer cells (M109) when co-cultured with anti-hapten antibody and human serum (M109_comp = folate glycosamine competitor). [Figure 45-1]Figures 45A and 45B show data relating to an ADCC assay plotted against FDH concentration (nM) versus ADCC kill percentage, showing Compound 150 administered to multiple mouse and human cancer cell lines in vitro (4T1-FR=Compound 150 and 4T1-FR_comp=Folate Glucosamine Competitor administered to 4T1 cells; MDA-MB-231=Compound 150 and MDA-MB-231_comp=Folate Glucosamine Competitor administered to MDA-MB-231 cells; M109=Compound 150 and M109_comp=Folate Glucosamine Competitor administered to M109 cells; THP-1=Compound 150 and THP-1_comp=Folate Glucosamine Competitor administered to THP-1 cells (a human monocytic leukemia cell line that serves as an in vitro model of acute myeloid leukemia (AML)). [Figure 45-2] Figures 45C and 45D show data relating to an ADCC assay plotted against FDH concentration (nM) versus ADCC kill percentage, showing Compound 150 administered to multiple mouse and human cancer cell lines in vitro (4T1-FR=Compound 150 and 4T1-FR_comp=Folate glucosamine competitor administered to 4T1 cells; MDA-MB-231=Compound 150 and MDA-MB-231_comp=Folate glucosamine competitor administered to MDA-MB-231 cells; M109=Compound 150 and M109_comp=Folate glucosamine competitor administered to M109 cells; THP-1=Compound 150 and THP-1_comp=Folate glucosamine competitor administered to THP-1 cells (a human monocytic leukemia cell line that serves as an in vitro model of acute myeloid leukemia (AML)). [Diagram 46]Shown are data related to an in vivo mouse lung cancer efficacy study of Compound 150 shown as a graph of tumor volume versus days after tumor implantation (PBS (+Abs) = control group; anti-PD1 + carboplatin + paclitaxel = SOC test article; and Compound 150 test article). Abs = antibody, in the case of the study shown in Figure 46, mouse anti-PD1 antibody (InVivoMAb anti-mouse PD-1 Catalog No. BE0146; InVivoGen, San Diego, CA). [Figure 47] 46 shows data related to an in vivo lung cancer efficacy study of Compound 150 shown in a graph of subject weight percent versus days after tumor implantation (PBS (+Abs) = control group; anti-PD1 + carboplatin + paclitaxel = SOC test article; and Fol-Dual Hapten (+Abs) = Compound 150 test article). Abs = anti-PD1 antibodies. [Figure 48] 1 shows data related to an in vivo human lung cancer efficacy study of Compound 150 shown in a graph of tumor volume versus days after tumor implantation (PBS (+Abs) = control group; anti-PD1 + radiotherapy = SOC test article; and Compound 150 test article). [Figure 49] Figure 48 shows safety data associated with an in vivo human lung cancer efficacy study of Compound 150, shown as a graph of subject body weight percentage versus days after tumor implantation (PBS (+Abs) = control group; anti-PDL1 + radiation therapy = SOC test article; and Compound 150 test article). [Figure 50] 1 shows data relating to an in vivo mouse colorectal cancer efficacy study of Compound 150 shown in a graph of tumor volume versus days after tumor implantation (PBS (+Abs) = control group; Leucovorin + 5-FU + Oxaliplatin = SOC test article; and Fol-Dual Hapten (+Abs) = Compound 150 test article). [Figure 51] Figure 50 shows safety data associated with an in vivo colorectal cancer efficacy study of Compound 150, shown as a graph of subject body weight percentage versus days after tumor implantation (PBS (+Abs) = control group; anti-PD1 + carboplatin + paclitaxel = SOC test article; and Compound 150 (+ABs) test article). [Figure 52]1 shows data relating to an in vivo mouse lung cancer combination therapy study shown in a graph of tumor volume versus days after tumor implantation. [Diagram 53] 1 shows data relating to an in vivo mouse lung cancer combination therapy study shown in a graph of tumor volume versus days after tumor implantation. [Figure 54] FIG. 1 shows data related to an in vivo mouse lung cancer combination therapy study shown in a graph of tumor volume versus days after tumor implantation (PBS (+Abs) = control group; Leacovorin+5FU+OXH = SOC test article; and Fol-Dual Hapten (+Abs) = Compound 150 test article). [Figure 55] Figure 54 shows data relating to an in vivo human lung cancer efficacy study of Compound 150, shown as a graph of subject body weight percentage versus days after tumor implantation (PBS (+Abs) = control group; Leacovorin + 5FU + OXH = SOC test article; and Fol-Dual Hapten (+Abs) = Compound 150 test article). [Figure 56] 1 shows data relating to an in vivo mouse colorectal cancer combination therapy study shown in a graph of tumor volume versus days after tumor implantation. [Figure 57] FIG. 1 shows data related to an in vivo mouse colorectal cancer combination therapy study depicted in a graph of tumor volume values ​​versus days after tumor implantation (PBS (+Abs) = control group; Leacovorin + 5FU + OXH = SOC test article; Fol-Dual Hapten (+Abs) = Compound 150 test article; Fol-Dual Hapten + Sunitinib = Compound 150 + Sunitinib; Fol-Dual Hapten + FA-TLR7 = Compound 150 + FA-TLR7). [Figure 58] 1 shows data relating to an in vivo mouse lung cancer comparative efficacy study of Compound 150 anti-PD1 + chemotherapy treatment shown in a graph of tumor volume versus days after tumor implantation (PBS (+Abs) = control group; Anti-PD1 + chemotherapy = SOC test article; and Fol-Dual Hapten (+Abs) = Compound 150 test article). [Figure 59] 1 shows data relating to an in vivo mouse lung cancer (cold) efficacy study of Compound 150 shown in a graph of tumor volume versus days after tumor implantation. [Figure 60] 1 shows data relating to an in vivo mouse kidney cancer (Renca cell line) efficacy study shown in a graph of tumor volume versus days after tumor implantation. [Figure 61] Graph showing comparative data of immune cell populations in Y856, LLC-1, and M109 cell lines (cold vs. hot) measured after treatment with PBS (control), Compound 150 (FDH), or SOC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] While the disclosure is susceptible to various modifications and alternative forms, exemplary embodiments thereof have been shown by way of example in the drawings and are herein described in detail.

[0057] Detailed Description Although the concepts of the present disclosure have been illustrated and described in detail herein, it is understood that the results herein are to be considered illustrative and not limiting in nature, that only exemplary embodiments have been shown and described, and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[0058] The present disclosure provides materials and methods for recruiting antibodies to the surface of viruses, virus-infected cells, cancer cells, immune cells, or fibroblasts, etc. See, for example, Figure 1A for a schematic illustration of antibody recruitment.

[0059] More specifically, a targeted therapeutic strategy is disclosed that has a dual mechanism of action to induce a host immune response against a target (e.g., a virus, a virus-infected cell, a cancer cell). The conjugate of the present invention can be trivalent, so that the conjugate comprises a targeting ligand linked (e.g., via a linker) to at least two haptens that bind to naturally occurring antibodies (e.g., antibodies in humans). Once recruited, these anti-hapten antibodies bind and activate the innate immune system against the target. Pharmaceutical compositions comprising such conjugates, as well as methods comprising administration of such conjugates and compositions, are also provided.

[0060] Conjugates The conjugate of the present invention can be a small molecule ligand-targeted drug conjugate that combines a receptor-specific ligand with at least two haptens. Thus, the conjugate can be a trivalent drug that can target desired cells with a receptor-specific ligand (e.g., a targeting ligand) and also bind to two or more antibodies in a subject with a dual payload. The overall scheme is to provide a specific targeting ligand conjugated to an effective payload of two or more haptens to treat viral infection or cancer. The targeting ligand can specifically recognize a target receptor (e.g., an envelope protein of a virus (which may only be expressed on the surface of infected cells) or a receptor that is overexpressed on a target cell (e.g., a folate receptor on a cancer cell, etc.)). In certain embodiments, one or more of the haptens are selected to activate the subject's innate immune system (e.g., adjacent to the target cell) to recruit immune cells and / or otherwise harness the subject's own immune system against the virus or cancer.

[0061] In certain embodiments, the conjugate has the formula: TL-LH n or a pharma- ceutically acceptable salt thereof; During the ceremony, TL is a targeting ligand for a target protein on the surface of a target cell, L is a linker, H is a hapten, n is an integer equal to or greater than 2. Optionally, at least two of H may be bound by different antibodies, e.g., when contacted with an antibody present in vivo. In certain embodiments, n is an integer equal to or greater than 2 (e.g., 2, 3, 4, 5, . . ., 10, etc.). n may be 2 or 3.

[0062] In many embodiments, the conjugate has the formula:

[0063] [ka] (including pharmaceutical salts thereof), where TL is a targeting ligand for a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell, or a fibroblast cell, and L a , L b , L c , and L d are each a linker, C is a carbon atom, and H1, H2, and H3 are each a hapten.

[0064] In these and other embodiments, at least two of the haptens are bound by different antibodies, for example when contacted with antibodies present in vivo. In these and other embodiments, H1 and H2 can each bind to a different antibody. In these and other embodiments, H1, H2, and H3 can each bind to a different antibody.

[0065] Optionally, the conjugate may be conjugated to an antibody via one or more of the haptens of the conjugate. When bound, each hapten is typically independently bound to a different antibody. Each antibody may be a naturally occurring autoantibody, an exogenously administered autoantibody, or an exogenously administered immunoglobulin G (IgG) antibody. Binding to the antibody is typically achieved by, for example, hydrogen bonding, coulombic bonding, Ca ++ Non-covalent interactions such as by cross-linking and Lifshitz-van der Waals bonds. This binding typically occurs in vivo.

[0066] Each of H, or H1, or H2, or H3 (if applicable) can be independently selected from different haptens. In certain embodiments (n=2), the haptens are different haptens. For n=3 or more, two or more of the haptens can be the same. Haptens that can be used include, but are not limited to, rhamnose fragments, α-galactosyl moieties, dinitrophenyl (DNP) fragments, trinitrophenyl (TNP) fragments, fluorescein fragments, digoxigenin fragments, biotin fragments, or antigens of diphtheria, varicella zoster virus, human papilloma virus (HPV), influenza virus antigens (e.g., hemagglutinin or neuraminidase), SARS-COV-2, yellow fever, respiratory syncytial virus (RSV), herpes simplex virus (HSV), varicella virus, A. Viral antigens selected from hepatitis (HAV) antigens (e.g., L-HbsAg, S-HbsAg, M-HbsAg, and preS), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV), hepatitis G (HGV), rotavirus, mumps virus, tetanus, human immunodeficiency virus (HIV) (e.g., gp120 or gp160), cytomegalovirus (CMV), vesicular stomatitis virus (e.g., glycoproteins), rubella virus, smallpox, monkeypox, poliovirus, dengue virus, and measles virus.

[0067] In some embodiments, the haptens are each independently selected from a rhamnose fragment, an α-galactosyl moiety, and a DNP fragment, In some embodiments, when n is 2, the haptens are a rhamnose fragment and a DNP fragment.

[0068] The hapten can be gp120 or gp160. The hapten can be a glycoprotein.

[0069] The targeting ligand of the conjugate can specifically recognize a target receptor (e.g., a target protein on the surface of a target cell), which can be a virus, a virus-infected cell, a cancer cell, an immune cell, or a fibroblast.

[0070] By specifically delivering haptens to cells recognized by the targeting ligand, the conjugates of the present invention can show high selectivity for malignant cells, virus-infected cells, immune cells, and any other cells that have target proteins on their surface, while also reducing the associated toxicity.To date, many cancers have been addressed by small molecule ligand-targeted drug conjugates that target receptors that are overexpressed on tumor cells.These overexpressed receptors include, but are not limited to, folate receptors (FR), prostate-specific membrane antigen (PSMA), cholecytokinin 2 receptors (CCK2R), carbonic anhydrase IX (CA IX), and the like.The targeting ligand of the present conjugates can have specificity for any of such overexpressed receptors.

[0071] The targeting ligand may also have specificity for a viral envelope protein, for example, the target protein may be an envelope protein of a virus or a viral envelope protein on the surface of a virus-infected cell.

[0072] For example, influenza viruses are enveloped viruses. All influenza subtypes are very similar in overall structure and organization, i.e., the virus particles are 80-120 nanometers in diameter and have a viral envelope containing two main types of glycoproteins that encase a central core. The central core contains the viral ribonucleic acid (RNA) genome and other viral proteins that package and protect this RNA. Unusually for a virus, the influenza genome is not a single nucleic acid, but rather seven or eight pieces of segmented negative-stranded RNA, each of which contains one or two genes that code for a gene product (protein). For example, the influenza A genome contains 11 genes on eight pieces of RNA that code for the following 11 proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1, M2, NS1, NS2 (NEP: nuclear export protein), PA, PB1 (polymerase basic 1), PB1-F2, and PB2.

[0073] HA and NA are two large glycoproteins on the outside of the virus particle. HA is a lectin that can mediate the binding of the virus to target cells and the entry of the viral genome into the target cells, and NA is typically involved in the release of progeny viruses from infected cells by cleaving the sugars that are attached to mature virus particles. The target protein can be influenza NA or influenza HA.

[0074] In some embodiments of the conjugate of the present invention, the targeting ligand is an NA inhibitor or an HA inhibitor, which can be used to deliver the conjugate to virus-infected cells and / or virus replication sites (e.g., nose, throat, and lungs).This allows virus-infected cells to be killed before the release of progeny viruses, to prevent virus replication, and / or to attenuate the early cytokine storm induced by virus infection.In certain embodiments, the targeting ligand is an oseltamivir fragment, a zanamivir fragment, a peramivir fragment, or a laninamivir fragment.In certain embodiments, the targeting ligand is zanamivir.

[0075] In other embodiments, the target protein is selected from respiratory syncytial virus (RSV) fusion protein F, coronavirus spike protein, hepatitis B virus (HBV) surface antigen or HBV core antigen, a cell surface receptor on a cancer cell, folate receptor alpha, prostate specific membrane antigen (PSMA), carbonic anhydrase 9 (CAIX), luteinizing hormone releasing hormone receptor (LHRH), neurokinin 1 receptor (NK1R), a cell surface receptor on tumor associated macrophages (TAM) or myeloid derived suppressor cells, folate receptor beta, a cell surface receptor on cancer associated fibroblasts, and fibroblast activation protein (FAP).

[0076] In certain embodiments, the targeting ligand is folate or an analog thereof (e.g., 5-methyltetrahydrofolate (5-MTHF). By "folate" is meant a folate receptor binding molecule, including, for example, folic acid, and analogs and derivatives of folic acid (e.g., but not limited to, folates, pteroylpolyglutamic acid, pteroyl-D-glutamic acid), and folate receptor binding pterdins (e.g., tetrahydropterin, dihydrofolic acid, tetrahydrofolic acid), and deaza and dideaza analogs thereof.

[0077] The terms "deaza" and "dideaza" analogs refer to art-recognized analogs in which one or two nitrogen atoms are replaced with carbon atoms in the naturally occurring folic acid structure, or analogs or derivatives thereof.For example, deaza analogs can include 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs of folate, folinic acid, pteropolyglutamic acid, and folate receptor binding pteridines (e.g., tetrahydropterin, dihydrofolic acid, and tetrahydrofolic acid).Dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs of folate. Other folates useful as complexing ligands in the context of the present disclosure include the folate receptor binding analogs pemetrexed, proguanil, pyrimethamine, trimethoprim, pralatrexate, raltitrexed, aminopterin, amethopterin (also known as methotrexate), N 10 -methylfolate, 2-deamino-dihydroxyfolate, deaza analogues such as 1-deazamethopterin or 3-deazamethopterin, and 3',5'-dichloro-4-amino-4-deoxy-N 10 -Methylpteroylglutamic acid (dichloromethotrexate).

[0078] Folic acid, as well as the analogs and / or derivatives described above, are also referred to as "a folate," "the folate," or "folates," reflecting their ability to bind to the folate receptor. As described herein, such molecules, when conjugated with exogenous molecules, are effective in enhancing transmembrane transport, for example, by folate-mediated endocytosis. The foregoing may be used in the folate receptor targeting ligands described herein.

[0079] In many embodiments, the conjugates include a linker (L) that couples or otherwise connects each hapten to a targeting ligand. In many embodiments, the conjugates include a linker L a , L b , L c In some embodiments, the conjugate comprises a linker, L a , L b , L c , and L d The conjugate comprises a plurality of linkers (e.g., L a , L b , L c , and optionally L d ), these linkers can be the same or different. For example, but not limited to, L a , L b , L c , and L d Each of L may have the same structure. Alternatively, one or more of these linkers may have a different structure compared to at least one of the other linkers. In certain embodiments, L a comprises a first structure, L b , L c , and L, if present d In certain embodiments, L b , L c , and L, if present d include structures that are different from each other.

[0080] As used herein, the term "linker" includes a chain of atoms that is biofunctionally adapted to form a chemical bond with a TL, and / or a hapten, and / or a carbon atom, and connects two or more functional moieties of a molecule to form a conjugate of the present invention. Illustratively, the chain of atoms can be selected from carbon (C), nitrogen (N), oxygen (O), sulfur (S), silicon (Si), and phosphorus (P). The chain of atoms can covalently connect various functional capabilities of the conjugate (e.g., targeting moieties and haptens). The linker can include a wide variety of links, such as ranging from about 2 to about 100 atoms in a contiguous backbone. The linker can include a time-release linker (e.g., a non-hydrolyzable linker).

[0081] In certain embodiments where the conjugate comprises two haptens, the conjugate may comprise at least three linkers: a first linker connects the targeting moiety to a carbon atom, a second linker connects the carbon atom to the first hapten, a third linker connects the carbon atom to the second hapten, and a second linker connects the targeting moiety to the second hapten, as shown in the haptens of formula IA and formula IB. Alternatively, the conjugate may comprise a single branched linker connecting all the haptens to the targeting moiety (see, for example, formula I). ​​In certain embodiments, the linker is a branched linker, and at least two of the haptens of the conjugate are connected to different branches of the linker. For example, the linker may comprise a backbone that comprises at least two branches (e.g., two branches, three branches, etc.) extending from the backbone. The different branches may comprise the same structure as the backbone and / or other branches, or may comprise different structures. The different branches can, for example, extend from different atoms of the backbone linker or can extend from the same atom (e.g., as shown in Formulas IA and IB).

[0082] In many embodiments, the linker comprises a polyethylene glycol (PEG) linker, a PEG derivative linker, a peptide, an alkyl group, a peptidoglycan, or a combination of two or more of the foregoing. In some embodiments, the PEG linker is (-CH2CH2-O-) n where n is an integer from 1 to 32, inclusive. In certain embodiments, the PEG linker's n is an integer from 1 to 16, inclusive. In certain embodiments, the linker is an optionally substituted heteroalkyl.

[0083] In some embodiments, the linker is a substituted heteroalkyl containing at least one substituent selected from the group consisting of alkyl, hydroxyl, oxo, PEG, carboxylate, and halo. "Halo" or "halogen," by itself or as part of another substituent, means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0084] In some embodiments, the linker comprises a spacer. In some embodiments, the spacer comprises a peptidoglycan or a sugar.

[0085] The linker is C2~C 18It may include alkyl groups, peptide fragments, or peptidoglycan fragments. As used herein, the term "fragment" refers to a molecule that has been modified to allow for linkage within a conjugate, either as a monovalent linkage, for example in the case of a targeting ligand or hapten, or as a bivalent linkage, for example in the case of a linker (L). For example, the term "zanamivir fragment" refers to a molecule that has been chemically modified to allow for zanamivir to be prepared as a targeting ligand covalently attached to a linker. For zanamivir, for example, a proton from an alcohol is missing, resulting in an open valence on the corresponding oxygen, which is then attached to the linker L. Typically, the alcohol oxygen closest to the dihydropyran ring of zanamivir forms a bond to the linker L. Similarly, a hapten fragment is a portion based on the corresponding molecule that is adapted for linkage to the linker L. For linker L, the corresponding fragment has an open valence to allow for linkage to other linker moieties, targeting ligands, and / or haptens. For example, for PEG, (-CH2CH2-O-) n The term "fragment" refers to a fragment that is attached on both sides to another moiety (e.g., another linker component). The use of the term "fragment" does not require that, from a synthetic standpoint, the molecule it refers to is made during the preparation of the conjugate. It is a description of the moiety within the conjugate, regardless of how it is made.

[0086] Optionally, the conjugate may be complexed with an antibody, for example when in vivo. Such complexation may be with a different antibody for each hapten.

[0087] In many embodiments of the present disclosure, the conjugate has Formula I:

[0088] [ka] or a pharma- ceutically acceptable salt thereof, wherein L1, L2, and L3 are each a linker. In certain embodiments, L1, L2, and L3 each independently comprise a PEG moiety, an alkyl group, a peptide group, and a peptidoglycan group. In certain embodiments, the PEG moiety (-CH2CH2-O-) n has n ranging from 1 to 16, inclusive. The alkyl group can be an alkyl chain of 2 to 18 carbons, inclusive (e.g., 18 carbons). For example, L1, L2, and L3 can each independently be C2 to C3 18 L1, L2, and L3 can each independently comprise a peptide or peptidoglycan fragment.

[0089] In certain embodiments, L1 comprises an amide, which may be optionally substituted with an alkyl group, a PEG moiety, an optionally substituted or unsubstituted triazole moiety (when substituted, the substitution may be a carbonyl alkyl group), and an amide, which may be optionally substituted with an alkyl group. Furthermore, in certain embodiments, the amide in L1 is directly bonded to the oxygen of the zanamivir fragment as described in formula I, forming a carbyl moiety with the oxygen. In certain embodiments, the tertiary carbon at the intersection of L1, L2, and L3 is bonded to the nitrogen of the amide of L1. In certain embodiments, the PEG moiety is bonded to the amide on one side and to the amide or substituted or unsubstituted triazole moiety on the other side. The PEG moiety is (-CH2CH2-O-) n where n can be 1 to 32, inclusive, 2 to 16, inclusive, 3 to 8, inclusive, and 4 to 7, inclusive. In some embodiments, n is 6.

[0090] In certain embodiments, L2 comprises an amide moiety, optionally substituted with an alkyl group, and a PEG moiety. The PEG moiety is (-CH2CH2-O-). nwhere n can generally be 1 to 32, inclusive, 2 to 16, inclusive, 3 to 8, inclusive, and 3 to 5, inclusive. In some embodiments, n is 4.

[0091] In certain embodiments, L3 comprises an alkylamide group, a carbonylalkyl group, an etheralkyl group, and an amine group that may be optionally substituted with an alkyl group. In some embodiments, L3 comprises PEG. In some embodiments, L3 comprises (-CH2CH2-O-) n wherein n can be 1 to 32, inclusive.

[0092] In one particular embodiment, compound 24:

[0093] [ka] or a pharma- ceutically acceptable salt thereof.

[0094] In one particular embodiment, compound 100:

[0095] [ka] or a pharma- ceutically acceptable salt thereof.

[0096] In certain embodiments, the conjugate comprises compound 101:

[0097] [ka] or a pharma- ceutically acceptable salt thereof, where Ac is acetate.

[0098] In certain embodiments, the conjugate comprises a folate targeting moiety. In certain embodiments, the conjugate is a folate dual hapten conjugate. In certain embodiments, the conjugate is a folate dual hapten conjugate, and is represented by compound 150:

[0099] [ka] or a pharma- ceutically acceptable salt thereof.

[0100] In some embodiments, one or more of the hydroxyl (-OH) groups in the structure of compounds 24, 100, 101, and / or 150 may be independently replaced with a thiol, a phosphate ester or a phosphonate ester, or -OC(=O)R1, where R1 is an alkyl group. In such embodiments, the amine (-NH2) group may be replaced with -OC(=O)R2, where R2 is an alkyl group. In these and other embodiments, the carboxyl (-COOH) group may be replaced with -OC(=O)R3, where R3 is an alkyl group. R2 and R3 may each be, for example, a C1-C6 alkyl group.

[0101] Optionally, the conjugate of the present invention (e.g., Compound 24, Compound 100, Compound 101, or Compound 150) is conjugated to an antibody in vivo. Such conjugation can be performed with a different antibody for each hapten.

[0102] In certain embodiments, a process for producing the conjugate of the present invention is provided.For example, the conjugate of compound 24 can be prepared as described in Example 1, and / or the conjugate of compound 150 can be prepared as described in Example 19.Similarly, other conjugates of the present invention can be prepared according to the process of Examples 1 and / or 19, as well as other such processes known in the art.

[0103] In certain embodiments, the conjugates are formulated as prodrugs. The term "prodrug" refers to a derivative of a conjugate that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to produce an active compound or conjugate, particularly the multi-hapten conjugates disclosed herein. Examples of prodrugs include, but are not limited to, derivatives and metabolites of the conjugates of the present invention that contain a biohydrolyzable moiety (e.g., biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogs). Specific prodrugs of compounds with a carboxyl functional group are lower alkyl esters of the carboxylic acid. Carboxylic acid esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule. Prodrugs can typically be prepared using known methods, for example, those described in Burger's Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers GmbH).

[0104] Formulation of the conjugate as a prodrug may incorporate protecting groups that may further retard hydrolysis of the conjugate in vivo, which may be beneficial when administering the prodrug to reprogram immune cells in a target area and / or to retard cytokine activation in the target area.

[0105] The compound and optional one or more other therapeutic agents can be administered as it is (unmodified) or in the form of pharmaceutically acceptable salt.When used in medicine, the salt should be pharmaceutically acceptable, but pharmaceutically unacceptable salts can be used to prepare pharmaceutically acceptable salts.Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid.Such salts can also be prepared as alkali metal salts or alkaline earth metal salts, for example, as sodium, potassium, or calcium salts of carboxylic acid group.

[0106] Suitable buffering agents include acetic acid and salts (1-2 w / v%), citric acid and salts (1-3 w / v%), boric acid and salts (0.5-2.5 w / v%), and phosphoric acid and salts (0.8-2 w / v%). Suitable preservatives include benzalkonium chloride (0.003-0.03 w / v%), chlorobutanol (0.3-0.9 w / v%), parabens (0.01-0.25 w / v%), and thimerosal (0.004-0.02 w / v%).

[0107] Salts (e.g., pharma- ceutically acceptable salts) of the compounds described herein may be prepared in a variety of ways, for example, by including an acid in the mobile phase during chromatographic purification or by stirring the product after chromatographic purification with an acid solution.

[0108] The conjugates of the present invention may be "deuterated", meaning that one or more hydrogen atoms may be replaced with deuterium. Since deuterium and hydrogen have nearly identical physical properties, deuterium replacement is the smallest structural change that can be made. Deuteration is known to those skilled in the art.

[0109] The conjugates may, in some embodiments, contain one or more asymmetric centers, thus resulting in enantiomers, diastereomers, and other stereoisomers defined in terms of absolute stereochemistry as (R)- or (S)-. In certain embodiments, the conjugates are in the R configuration. In certain embodiments, the conjugates are in the S configuration. Unless otherwise stated, all stereoisomers of the conjugates are intended to be contemplated. In cases where the conjugates contain an alkene double bond, both E and Z geometric isomers (e.g., cis or trans) and / or optical isomers are intended to be included unless otherwise specified. In certain embodiments, for example, D and A of the conjugate are arranged in a relative cis orientation. In certain embodiments, D and A of the conjugate are arranged in a relative trans orientation. Similarly, all possible isomers, as well as their racemic and optically pure forms, and all tautomers are also intended to be included. The term "geometric isomer" refers to the E or Z geometric isomers (e.g., cis or trans) of an alkene double bond. The term "positional isomer" refers to structural isomers about a central ring, e.g., ortho, meta, and para isomers about a benzene ring.

[0110] Furthermore, in each of the above and below embodiments, the formula should be understood to include and represent all pharma- ceutically acceptable salts of the conjugate, as well as any hydrates and / or solvates of the conjugate formula or its salts.Indeed, hydrates, solvates, and N-oxides of the conjugate are also contemplated.The term "solvate" refers to a conjugate or its salt that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces.When the solvent is water, the solvate is a hydrate.

[0111] It will be understood that certain functional groups (e.g., hydroxy, amino, and similar groups) will form complexes and / or coordination compounds with water and / or various solvents in the various physical forms of the conjugates, and therefore the above formulas should be understood to include and represent these various hydrates and / or solvates.

[0112] In each of the above and below embodiments, the formulas should also be understood to include and represent all crystalline, partially crystalline, and non-crystalline and / or amorphous forms of the conjugate.

[0113] Pharmaceutical Compositions Further, a pharmaceutical composition is provided. The pharmaceutical composition may comprise any of the conjugates described herein (e.g., the conjugates of formula I) or their pharma- ceutically acceptable salts, and one or more pharma-ceutically acceptable excipients or carriers. The term "composition" generally refers to any product that contains multiple components, such as the conjugates. The composition may be prepared from isolated conjugates, or salts, solutions, hydrates, solvates, and other forms of the conjugates.

[0114] In certain embodiments, the pharmaceutical composition comprises a plurality of conjugates (e.g., two or more) and a pharma- ceutically acceptable excipient. In certain embodiments, the pharmaceutical composition further comprises at least one additional pharma- ceutically active agent. The at least one additional pharma- ceutically active agent may be an agent useful in the treatment of viral infection, fibrosis, or cancer.

[0115] Pharmaceutical compositions may be prepared by combining one or more conjugates, a pharma- ceutically acceptable excipient, and, optionally, one or more additional pharma- ceutical active agents, according to methods known in the art and described herein below.

[0116] The pharmaceutical composition of the present invention may comprise one or more pharma- ceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles (e.g., conventional non-toxic pharma- ceutically acceptable carriers, adjuvants, and vehicles), and combinations thereof. Any pharma- ceutically acceptable carriers, diluents, and excipients known in the art may be used. Examples include, but are not limited to, excipients, colorants, preservatives, and stabilizers. More specific examples include crystalline cellulose, carmellose calcium, carmellose sodium, hydropropyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose, and magnesium stearate.

[0117] Solutions of the conjugate or pharmaceutical composition may be aqueous, optionally mixed with non-toxic surfactants and / or contain carriers or excipients such as salts, carbohydrates, and buffers (preferably pH 3-9), but may be more suitably formulated as sterile non-aqueous solutions or as dry forms to be used in combination with a suitable vehicle such as sterile pyrogen-free water or phosphate-buffered saline, depending on the application. For example, dispersions may be prepared in glycerol, liquid PEG, triacetin, and mixtures thereof, as well as oils. Under ordinary conditions of storage and use, these preparations may further contain a preservative to prevent the growth of microorganisms.

[0118] The conjugates can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to a selected route of administration. The pharmaceutical compositions can, for example, be formulated for a given route of administration and can be administered in accordance with methods in the art and in, for example, Remington, The Science and Practice of Pharmacy, 22 nd The composition may be prepared according to the methods described in the 2012 edition of the American Pharmaceutical Association (APA). The composition may be an injectable or injectable composition, such as a composition that may be injected subcutaneously or intravenously.

[0119] The pharmaceutical composition may be administered to a mammalian host, such as a human patient, in a variety of forms adapted to the selected route of administration. In certain embodiments, the pharmaceutical composition is formulated to be administered subcutaneously. In certain embodiments, the pharmaceutical composition is formulated to be administered orally. In certain embodiments, the pharmaceutical composition is formulated to be administered intramuscularly, intravenously, intraarterially, intraperitoneally, or any other parenteral administration recognized in the art.

[0120] The pharmaceutical composition may be administered systemically in combination with a pharma- ceutically acceptable vehicle. The percentage of the components of the composition and preparation may vary and may be about 1 to about 99% by weight of the active ingredient (e.g., the compound or the conjugate) and binders, excipients, disintegrants, lubricants, and / or sweeteners (known in the art). The amount of active conjugate in such therapeutically useful compositions is that which allows for obtaining an effective dosage level (e.g., in serum or target tissues or cells).

[0121] Exemplary means of parenteral administration include needle (including microneedle) injectors, needle-free injectors, and injection techniques, as well as any other means of parenteral administration recognized in the art. Parenteral formulations are typically aqueous solutions, which may contain excipients such as salts, carbohydrates, and buffers (preferably at a pH ranging from about 3 to about 9), although for some applications they may be more suitably formulated as sterile non-aqueous solutions or as dry forms used in combination with a suitable vehicle, such as sterile pyrogen-free water. Preparation of parenteral formulations under sterile conditions, for example by lyophilization, may be readily accomplished using standard pharmaceutical techniques known to those skilled in the art.

[0122] Pharmaceutical dosage forms suitable for administration may include sterile aqueous solutions or dispersions, or sterile powders, containing the active ingredient suitable for extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes, nanocrystals, or polymer nanoparticles. In any case, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or medium may be, for example, a solvent or liquid dispersion medium, including, but not limited to, water, electrolytes, sugars, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and / or suitable mixtures thereof. In at least one embodiment, the desired fluidity may be maintained by the formation of liposomes, or by maintaining the required particle size in the case of dispersions, or by the use of surfactants.

[0123] Sterile injectable solutions can be prepared by incorporating the pharmaceutical composition in the required amount of a suitable solvent and one or more of the other components described above, as required, followed by filter sterilization.For the preparation of sterile powder for sterile injectable solutions, vacuum drying and freeze-drying techniques can be adopted, which can obtain the powder of active ingredient and any additional desired ingredient that are present in the solution previously sterile-filtered.

[0124] method Further, in view of the above, a method of treating a viral infection in a subject is also provided. The method may include administering to the subject an effective amount of the conjugate, prodrug, or composition described above and a pharma- ceutically acceptable excipient (e.g., orally or intravenously (about 0.01 μg / kg to about 10 μg / kg of the conjugate). In certain embodiments, the "effective amount" is an amount effective to treat a viral infection, such as influenza. The method may further include administering an autoantibody or an allogeneic immunoglobulin G (IgG) antibody.

[0125] The viral infection can be influenza. The viral infection can be influenza A. The viral infection can be influenza B. The method can elicit an immune response that eliminates the Ab-coated virus, or cells infected with the Ab-coated virus, by antibody (Ab)-dependent cellular phagocytosis (ADCP), Ab-dependent cell-mediated cytotoxicity (ADCC), and / or complement-dependent cytotoxicity (CDC).

[0126] In view of the above, a method for treating cancer in a subject is also provided.The method comprises administering to the subject an effective amount of the above-mentioned conjugate, prodrug or composition and a pharma- ceutically acceptable excipient.The method can further comprise administering autoantibody, allogeneic IgG antibody or IVIG.

[0127] These cancers include lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, epithelial cancer, leiomyosarcoma, rectal cancer, stomach cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, The cancer may be small cell lung cancer, adrenal gland cancer, sarcoma of soft tissue, urethral cancer, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, pleural mesothelioma, bladder cancer, gastric cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, carcinoma of the renal pelvis, tumor of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, cholangiocarcinoma, Hürthle cell thyroid cancer, or adenocarcinoma of the gastroesophageal junction. The cancer may be lung cancer, triple negative breast cancer, colon cancer, gastric cancer, bladder cancer, prostate cancer, or pancreatic cancer.

[0128] In certain embodiments, the cancer is a hot cancer. Cancers that are refractory to checkpoint inhibitor treatment and typically have low immune infiltration are often referred to as "cold" cancers. Conversely, cancers or tumors that respond to treatment are often referred to as "hot" cancers, and typically have receptors on the cancer cell surface that can trigger a strong immune response in subjects. The hot cancer can be kidney cancer. The hot cancer can be lung cancer. The hot cancer can be colorectal cancer.

[0129] Combination therapy is also provided. In certain embodiments of the method of treating viral infection or cancer, the method further comprises administering a second treatment to the subject. In the case of the method of treating cancer, the second treatment can comprise administering a chemotherapeutic agent (e.g., an effective amount of a chemotherapeutic agent). The second treatment can comprise administering sunitinib (e.g., an effective amount of sunitinib). The second treatment can comprise administering a PD-1 inhibitor (e.g., an effective amount of a PD-1 inhibitor). The second treatment can comprise administering a PDL-1 inhibitor (e.g., an effective amount of a PDL-1 inhibitor).

[0130] The second treatment may include administration of a folate toll-like receptor 7 (FA-TLR7) agonist conjugate (e.g., an effective amount of a FA-TLR7 agonist conjugate). The FA-TLR7 agonist may act synergistically with compound 150 to further reduce tumor growth, as the FA-TLR7 agonist may reprogram activated macrophages in the tumor microenvironment (TME) by converting tumor-promoting macrophages into antitumor macrophages.

[0131] In certain embodiments, the second treatment comprises one or more of chemotherapy, radiation therapy, sunitinib, a PD-1 inhibitor, or a PDL-1 inhibitor. In certain embodiments, the method further comprises imaging the subject (e.g., using a known imaging modality) to obtain an image of the cancer (e.g., the cancerous tumor).

[0132] Also provided is a method for treating fibrosis in a subject. The method comprises administering to the subject an effective amount of the conjugate, prodrug or composition described above and a pharma- ceutically acceptable excipient. The method can further comprise administering an autoantibody, an allo-IgG antibody, or an IVIG.

[0133] Also provided is a method of activating an immune response in a subject (e.g., in a target region of a subject, such as the TME). In certain embodiments, the method of activating an immune response in a subject comprises administering to the subject an effective amount of the conjugate, prodrug, or composition described above, and a pharma- ceutically acceptable excipient. The immune response can be an innate immune response. The immune response can be activated at a target region of a subject, such as the TME, or at the location of a viral replication site (e.g., proximal to one or more virally infected cells). In the case where a prodrug is administered, such a prodrug can further delay hydrolysis of the underlying conjugate and / or delay cytokine activation in the target region of a subject.

[0134] The method of activating an immune response may further comprise administering to the subject an autoantibody, an allogeneic IgG antibody, or human IVIG.

[0135] Administration of an effective amount of the conjugate or composition can induce reprogramming of M2 macrophages to M1 macrophages in the target area. In general, and without any intended limitation, the novel conjugates, compositions, and methods of the present invention can target the innate immune system of a subject and reprogram the polarization of macrophages from M2 to M1 in favor of the pro-inflammatory nature of the M1 phenotype. For example, in at least one exemplary embodiment, such conjugates and compositions include a targeting moiety that targets a folate receptor (e.g., FRβ), conjugated to two or more haptens via a linker, and includes, for example, a folate receptor binding ligand, or an analog, functional fragment, derivative, or radical thereof (e.g., pteroylamino acid). Such embodiments take advantage of the restricted expression of folate receptors to directly localize systemically administered conjugates and compositions to folate-expressing cells (e.g., folate-expressing cells in cancer tissues), so that the haptenic component can then bind to the desired antibody, recruit immune cells to the target site, and reprogram activated myeloid cells (e.g., M2-like macrophages) to pro-inflammatory M1 polarization. This targeting design advantageously prevents systemic activation of the immune system (i.e., reduces systemic exposure to such conjugates / compositions), thus avoiding toxicity, and further promoting activation of the subject's own immune system at or near the target site. As previously mentioned, in certain embodiments, the conjugates or compositions of the present invention may be administered with a second treatment comprising administering an effective amount of a FA-TLR7 agonist, which may function synergistically with the conjugates / compositions of the present invention by further promoting the conversion of activated macrophages to anti-tumor (M1-like) macrophages.

[0136] The terms "treat," "treating," "treated," or "treatment" (with respect to a disease or condition) refer to an approach for obtaining beneficial or desired results, preferably including clinical results, including, but not limited to, one or more of the following: amelioration of conditions associated with the disease, curing the disease, reducing the severity of the disease, slowing the progression of the disease, alleviating one or more symptoms associated with the disease, improving the quality of life of a person suffering from the disease, extending survival, and / or prophylactic or preventative treatment.

[0137] "Effective amount" and "amount effective to treat" refer to any amount sufficient to achieve a desired biological effect. By selecting from among various active conjugates and considering factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and mode of administration, in combination with the teachings provided herein, one can design an effective prophylactic or therapeutic treatment regimen that causes little to no undesirable toxicity and is effective to treat a particular subject. The effective amount for any particular application may vary depending on factors such as the infection, cancer, or other condition being treated, the particular conjugate or composition being administered, concurrent or sequential treatments being administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art can empirically determine the effective amount of a particular compound and / or other therapeutic agent without necessitating undue experimentation. Maximum doses (i.e., the highest safe dose according to some medical judgment) may be used. Multiple doses per day may be used to achieve an appropriate systemic level of the compound. The appropriate systemic level may be determined, for example, by measuring the patient's peak or sustained plasma levels of the drug. "Dose" and "administration" are used interchangeably herein.

[0138] In general, the daily oral dose of the compound is about 0.01 milligrams / kg per day to 1,000 milligrams / kg per day for human subjects. Oral doses in the range of 0.5 to 50 milligrams / kg per day, in one or multiple administrations, can provide therapeutic results. The dosage can be adjusted appropriately to achieve the desired drug levels locally or systemically, depending on the mode of administration. For example, intravenous administration can vary from one to several orders of magnitude lower per day. If the subject does not respond adequately to such doses, higher doses (or higher effective doses by a different, more localized route of delivery) can be employed, as long as the patient tolerates them. Multiple doses per day (e.g., two doses per day) are contemplated to achieve adequate systemic levels of the conjugate.

[0139] An "effective amount" (or "amount effective to treat (therapeutically)") of a conjugate (used interchangeably herein as "compound") for use in treatment refers to the amount of compound in a preparation that, when administered as part of a desired dosing regimen (to a mammal such as a human), alleviates the symptoms, relieves the condition, or delays the onset of a disease state, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment, according to clinically accepted criteria for the disorder or condition being treated, or for cosmetic purposes.

[0140] For any conjugate, therapeutically effective amount can be determined first from animal models.Therapeutically effective amount can also be determined from human data for compounds that are being tested in humans and compounds known to exhibit similar pharmacological activity, such as other related active agents.For parenteral administration, higher doses may be required.Applied doses can be adjusted based on the relative bioavailability and efficacy of the administered compound.It is well within the capabilities of a person skilled in the art to adjust doses to achieve maximum efficacy based on the methods described above and other methods known in the art.

[0141] For clinical use, any compound may be administered in an amount equal to or equivalent to 0.2-2,000 milligrams (mg) of compound per kilogram (kg) of subject's body weight per day. The compound may be administered in a dose equal to or equivalent to 2-2,000 mg of compound per kg of subject's body weight per day. The compound may be administered in a dose equal to or equivalent to 20-2,000 mg of compound per kg of subject's body weight per day. The compound may be administered in a dose equal to or equivalent to 50-2,000 mg of compound per kg of subject's body weight per day. The compound may be administered in a dose equal to or equivalent to 100-2,000 mg of compound per kg of subject's body weight per day. The compound may be administered in a dose equal to or equivalent to 200-2,000 mg of compound per kg of subject's body weight per day. If a precursor or prodrug of a compound is to be administered, it is administered in an amount equivalent to the above amounts of the compound (ie, an amount sufficient to deliver the compound).

[0142] Formulations of the compounds may be administered to human subjects in therapeutically effective amounts. Typical dose ranges are 0.01 micrograms / kg to about 2 mg / kg body weight per day. The dosage of the drug administered will likely depend on variables such as the type and extent of the disorder, the overall health of the particular subject, the specific compound administered, the excipients used to formulate the compound, and its route of administration. Routine experimentation may be used to optimize the dosage and frequency of administration of any particular compound.

[0143] The conjugates may be administered at concentrations ranging from about 0.001 micrograms / kg to greater than about 500 mg / kg. For example, the concentrations may be 0.001 micrograms / kg, 0.01 micrograms / kg, 0.05 micrograms / kg, 0.1 micrograms / kg, 0.5 micrograms / kg, 1.0 micrograms / kg, 10.0 micrograms / kg, 50.0 micrograms / kg, 100.0 micrograms / kg, 500 micrograms / kg, 1.0 mg / kg, 5.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, 20.0 mg / kg, 25.0 mg / kg, 30.0 mg / kg, 35.0 mg / kg, 40.0 mg / kg, 50.0 mg / kg, 60.0 mg / kg, 70.0 mg / kg, 80.0 mg / kg, 90.0 mg / kg, 100.0 mg / kg, 120.0 mg / kg, 140.0 mg / kg, 160.0 mg / kg, 180.0 mg / kg, 190.0 mg / kg, 210.0 mg / kg, 220.0 mg / kg, 230.0 mg / kg, 240.0 mg / kg, 250.0 mg / kg, 260.0 mg / kg, 270.0 mg / kg, 280.0 mg / kg, 290.0 mg / kg, 300.0 mg / kg, 310.0 mg / kg, 320.0 mg / kg, 330.0 mg / kg, 340.0 mg / kg, 350.0 mg / kg, 360.0 mg / kg, 370.0 mg / kg, 380.0 g, 30.0 mg / kg, 35.0 mg / kg, 40.0 mg / kg, 45.0 mg / kg, 50.0 mg / kg, 60.0 mg / kg, 70.0 mg / kg, 80.0 mg / kg, 90.0 mg / kg, 100.0 mg / kg, 150.0 mg / kg, 200.0 mg / kg, 250.0 mg / kg, 300.0 mg / kg, 350.0 mg / kg, 400.0 mg / kg, 450.0 mg / kg, up to about 500.0 mg / kg or any increment therein. It should be understood that all values ​​and ranges between these values ​​and ranges are intended to be encompassed.

[0144] The conjugate may be administered at a dosage ranging from about 0.2 milligrams / kg / day to greater than about 100 mg / kg / day. For example, the dosage may range from 0.2 mg / kg / day to 100 mg / kg / day, 0.2 mg / kg / day to 50 mg / kg / day, 0.2 mg / kg / day to 25 mg / kg / day, 0.2 mg / kg / day to 10 mg / kg / day, 0.2 mg / kg / day to 7.5 mg / kg / day, 0.2 mg / kg / day to 5 mg / kg / day, 0.25 mg / kg / day to 100 mg / kg / day, 0.25 mg / kg / day to 50 mg / kg / day, 0.25 mg / kg / day to 25 ... day~10mg / kg / day, 0.25mg / kg / day~7.5mg / kg / day, 0.25mg / kg / day~5mg / kg / day, 0.5mg / kg / day~50mg / kg / day, 0.5mg / kg / day~25mg / kg / day, 0.5mg / kg / day Day~20mg / kg / day, 0.5mg / kg / day~15mg / kg / day, 0.5mg / kg / day~10mg / kg / day, 0.5mg / kg / day~7.5mg / kg / day, 0.5mg / kg / day~5mg / kg / day, 0.75mg / kg / day~ 50mg / kg / day, 0.75mg / kg / day ~ 25mg / kg / day, 0.75mg / kg / day ~ 20mg / kg / day, 0.75mg / kg / day ~ 15mg / kg / day, 0.75mg / kg / day ~ 10mg / kg / day, 0.75mg / kg / day~7.5mg / kg / day, 0.75mg / kg / day~5mg / kg / day, 1.0mg / kg / day~50mg / kg / day, 1.0mg / kg / day~25mg / kg / day, 1.0mg / kg / day~20mg / kg / day, 1.0mg / kg / day It can be 2mg / kg / day to 50mg / kg / day, 2mg / kg / day to 25mg / kg / day, 2mg / kg / day to 20mg / kg / day, 2mg / kg / day to 15mg / kg / day, 2mg / kg / day to 10mg / kg / day, 2mg / kg / day to 7.5mg / kg / day, or 2mg / kg / day to 5mg / kg / day.

[0145] The conjugate may be administered at a dosage ranging from about 0.25 milligrams / kg / day to about 25 mg / kg / day. For example, the dosage may be 0.25 mg / kg / day, 0.5 mg / kg / day, 0.75 mg / kg / day, 1.0 mg / kg / day, 1.25 mg / kg / day, 1.5 mg / kg / day, 1.75 mg / kg / day, 2.0 mg / kg / day, 2.25 mg / kg / day, 2.5 mg / kg / day, 2.75 mg / kg / day, 3.0 mg / kg / day, 3.25 mg / kg / day, 3.5 mg / kg / day, 3.75 mg / kg / day, 4.0 mg / kg / day, 5.0 mg / kg / day, 6.0 mg / kg / day, 7.0 mg / kg / day, 8.0 mg / kg / day, 9.0 mg / kg / day, 10.0 mg / kg / day, 11.0 mg / kg / day, 12.0 mg / kg / day, 13.0 mg / kg / day, 14.0 mg / kg / day, 15.0 mg / kg / day, 16.0 mg / kg / day, 17.0 mg / kg / day, 18.0 mg / kg / day, 19.0 mg / kg / day, 20.0 mg / kg / day, 21.0 mg / kg / day, 22.0 mg / kg / day, 23.0 mg / kg / day, 24.0 mg / kg / day, 25.0 mg / kg / day, 26.0 mg / kg / day, 27.0 mg / kg / day, 28.0 mg / kg / day, 29.0 mg / kg / day, 30.0 mg / kg / day, 31.0 mg / kg / day, 32.0 mg / kg / kg / day, 4.25mg / kg / day, 4.5mg / kg / day, 4.75mg / kg / day, 5mg / kg / day, 5.5mg / kg / day, 6.0mg / kg / day, 6.5mg / kg / day, 7.0mg / kg / day, 7.5mg / kg / day, 8.0mg / kg / day, 8.5mg / kg / day, 9.0mg / kg / day, 9.5mg / kg / day, 10mg / kg / day, 11mg / kg / day, 12mg / kg / day, 13mg / kg / day, 14mg / kg / day, 15mg / kg / day, 16mg / kg / day, 17mg / kg / day, 18mg / kg / day, 19mg / kg / day, 20mg / kg / day, 21mg / kg / day, 22mg / kg / day, 23mg / kg / day, 24mg / kg / day, 25mg / kg / day, 26mg / kg / day, 27mg / kg / day, 28mg / kg / day, 29mg / kg / day, 30mg / kg / day, 31mg / kg / day, 32mg / kg / day , 33 mg / kg / day, 34 mg / kg / day, 35 mg / kg / day, 36 mg / kg / day, 37 mg / kg / day, 38 mg / kg / day, 39 mg / kg / day, 40 mg / kg / day, 41 mg / kg / day, 42 mg / kg / day, 43 mg / kg / day, 44 mg / kg / day, 45 mg / kg / day, 46 mg / kg / day, 47 mg / kg / day, 48 mg / kg / day, 49 mg / kg / day, or 50 mg / kg / day.

[0146] The conjugate or precursor thereof may be administered at a concentration ranging from 0.01 micromolar to 500 micromolar or more. For example, the dose may be 0.01 micromolar, 0.02 micromolar, 0.05 micromolar, 0.1 micromolar, 0.15 micromolar, 0.2 micromolar, 0.5 micromolar, 0.7 micromolar, 1.0 micromolar, 3.0 micromolar, 5.0 micromolar, 7.0 micromolar, 10.0 micromolar, 15.0 micromolar, 20.0 micromolar, 25.0 micromolar, 30.0 micromolar, 35.0 micromolar, 40 micromolar, 50 micromolar, 60 micromolar, 70 micromolar, 80 micromolar, 90 micromolar, 100 micromolar, 110 micromolar, 120 micromolar, 130 micromolar, 140 micromolar, 150 micromolar, 160 micromolar, 170 micromolar, 180 micromolar, 190 micromolar, 210 micromolar, 220 micromolar, 230 micromolar, 240 micromolar, 250 micromolar, 260 micromolar, 270 micromolar, 280 micromolar, 290 micromolar, 300 micromolar, 300 micromolar, 350 micromolar, 360 micromolar, 370 micromolar, 380 micromolar, 390 micromolar, 400 micromolar, 410 micromolar, 420 micromolar, 430 micromolar, 440 micromolar, 450 micromolar, 460 micromolar, 470 micromolar, 4 The concentration may be 0.0 micromolar, 45.0 micromolar, 50.0 micromolar, 60.0 micromolar, 70.0 micromolar, 80.0 micromolar, 90.0 micromolar, 100.0 micromolar, 150.0 micromolar, 200.0 micromolar, 250.0 micromolar, 300.0 micromolar, 350.0 micromolar, 400.0 micromolar, 450.0 micromolar, up to about greater than 500.0 micromolar, or any increment therein. It should be understood that all values ​​and ranges between these values ​​and ranges are intended to be encompassed.

[0147] The conjugate or precursor thereof may be administered at a concentration ranging from 0.10 micrograms / mL to 500.0 micrograms / mL. For example, the concentrations may be 0.10 micrograms / mL, 0.50 micrograms / mL, 1 micrograms / mL, 2.0 micrograms / mL, 5.0 micrograms / mL, 10.0 micrograms / mL, 20 micrograms / mL, 25 micrograms / mL, 30 micrograms / mL, 35 micrograms / mL, 40 micrograms / mL, 45 micrograms / mL, 50 micrograms / mL, 60.0 micrograms / mL, 70.0 micrograms / mL, 80.0 micrograms / mL, 90.0 micrograms / mL, 100.0 micrograms / mL, 110.0 micrograms / mL, 120.0 micrograms / mL, 130.0 micrograms / mL, 140.0 micrograms / mL, 150.0 micrograms / mL, 160.0 micrograms / mL, 170.0 micrograms / mL, 180.0 micrograms / mL, 190.0 micrograms / mL, 200.0 micrograms / mL, 210.0 micrograms / mL, 220.0 micrograms / mL, 230.0 micrograms / mL, 240.0 micrograms / mL, 250.0 micrograms / mL, 260.0 micrograms / mL, 270.0 micrograms / mL, 280.0 micrograms / mL, 290.0 micrograms / mL, 300.0 micrograms / mL, 350.0 micrograms / mL, 400.0 micrograms / mL, 400.0 micrograms / mL, 400.0 micrograms / The amount of the glycerol in the solution may be 80.0 micrograms / mL, 90.0 micrograms / mL, 100.0 micrograms / mL, 150.0 micrograms / mL, 200.0 micrograms / mL, 250.0 micrograms / mL, 250.0 micrograms / mL, 300.0 micrograms / mL, 350.0 micrograms / mL, 400.0 micrograms / mL, 450.0 micrograms / mL, up to about greater than 500.0 micrograms / mL, or any increment thereof. It should be understood that all values ​​and ranges between these values ​​and ranges are intended to be encompassed.

[0148] The formulation may be administered in a pharmaceutically acceptable solution, which may always contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.When used in therapy, an effective amount of the conjugate may be administered to a subject in any mode that delivers the conjugate to the desired surface.Administering the pharmaceutical composition may be accomplished by any means known to those skilled in the art.Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (bladder), oral, subcutaneous, direct injection (e.g., direct injection into tumor or abscess), mucosa (e.g., topical to eye), inhalation, and topical.

[0149] For oral administration, the conjugate can be easily formulated by combining the active conjugate with pharma- ceutically acceptable excipients known in the art. Such excipients allow the conjugate to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by the subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipients, and optionally the mixture obtained is ground, and after adding suitable auxiliary agents as necessary, the mixture of granules is processed to obtain a tablet or dragee core. Suitable excipients include: fillers such as sugars, for example, lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP), etc. If necessary, disintegrating agents may be added, such as cross-linked PVP, agar, or alginic acid or a salt thereof (e.g., sodium alginate). Optionally, the oral formulations may also be formulated with saline or buffers (e.g., EDTA) to neutralize internal acidic conditions, or may be administered without excipients.

[0150] Also contemplated is an oral dosage form of the conjugate. The conjugate may be chemically modified to allow for effective oral delivery of the derivative. In general, the contemplated chemical modification is the addition of at least one moiety to the compound itself, which (a) inhibits acid hydrolysis, and (b) allows for uptake from the stomach or intestine into the bloodstream. Also desired is an increase in the overall stability of the compound and an increase in circulation time in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, PVP, and polyproline. Abuchowski and Davis, "Soluble Polymer-Enzyme Adducts," In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383 (1981); Newmark et al., Preparation and properties of adducts of streptokinase and streptokinase-plasmin complex with polyethylene glycol and pluronic polyol F38, J Appl Biochem 4: 185-189 (1982). Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. For pharmaceutical use, the polyethylene glycol moiety is preferred, as indicated above.

[0151] The release location of the compound of the present invention can be stomach, small intestine (for example, duodenum, jejunum, or ileum), or large intestine.Those skilled in the art can utilize formulations that do not dissolve in the stomach but release material in the duodenum or other places in the intestine.This release can avoid the harmful effects of the stomach environment by protecting the compound or releasing the compound beyond the stomach environment, for example, in the intestine.

[0152] To ensure full gastric resistance, coatings that are impermeable to at least pH 5.0 are typically utilized. Examples of the more common inactive ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings may be used as mixed films.

[0153] Tablets may also use a coating or mixture of coatings that are not intended to protect from the stomach. This may include sugar coatings, or coatings to make the tablet easier to swallow. Capsules may consist of a hard shell (e.g., gelatin) to deliver the dry therapeutic agent (e.g., powder), or in the case of liquid forms, a soft gelatin shell may be used. The shell material of cachets may be thick starch or other edible paper. For pills, lozenges, molded tablets, or tablet triturates, wet massing techniques may be used.

[0154] The conjugate may be included in the formulation as fine multiparticulates in the form of granules or pellets with a particle size of about 1 mm. The material for capsule administration may also be formulated as a powder, lightly compressed plugs, or tablets. The therapeutic agent may be prepared by compression.

[0155] Colorants and flavoring agents may all be included. For example, the compounds may be formulated (such as by liposome or microsphere encapsulation) and then further included in an edible product, such as a refrigerated beverage, that contains colorants and flavoring agents.

[0156] The compound can be diluted or its amount can be increased with inert material.The diluent can include carbohydrates, particularly mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextran, and starch.Certain inorganic salts such as calcium triphosphate, magnesium carbonate, sodium chloride, etc. can also be used as fillers.Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress, and Avicell.

[0157] In the formulation of the therapeutic agent into a solid dosage form, a disintegrant may be included. Substances used as disintegrants include, but are not limited to, starch (e.g., Explotab, a starch-based commercial disintegrant). Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge, and bentonite may all be used. Another form of disintegrant is the insoluble cation exchange resin. Powdered gums may be used as disintegrants and binders, and these may include powdered gums such as agar, Karaya, or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

[0158] Binders can be used to bind the compounds together to form hard tablets, and can include materials from natural products such as acacia, tragacanth, starch, and gelatin. Others include methylcellulose (MC), ethylcellulose (EC), and carboxymethylcellulose (CMC). Both PVP and HPMC can be used in alcoholic solutions to granulate the therapeutic agent.

[0159] Antifriction agents can be included in the formulation of therapeutic agent to prevent sticking during the formulation process.Lubricants can be used as a layer between therapeutic agent and die wall, and can include, but are not limited to, stearic acid (including its magnesium and calcium salts), polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oil, and wax.Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, Carbowax 4000 and 6000 can also be used.

[0160] Glidants may be added that may improve the flowability of the drug during formulation and aid in rearrangement during compression. Glidants may include starch, talc, pyrogenic silica, and hydrated silicoaluminate.

[0161] Surfactants may be added as wetting agents to facilitate dissolution of therapeutic agents in aqueous environments. Surfactants may include anionic detergents such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents that may be used include benzalkonium chloride and benzethonium chloride. Potential nonionic detergents that may be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, polysorbate 40, 60, 65, and 80, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. These surfactants may be present alone or as a mixture of various ratios in the formulation of the compound or its derivatives.

[0162] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin and sealed soft capsules made of gelatin and plasticizers such as glycerol or sorbitol. Push-fit capsules can contain active ingredients in a mixture with filler such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, active compounds can be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers can be added. Microspheres formulated for oral administration can also be used. Such microspheres are well defined in the art. All formulations for oral administration should be in a dosage suitable for such administration.

[0163] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0164] For administration by inhalation, the compound can be conveniently delivered in the form of aerosol spray presentation from pressurized pack or nebulizer using suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.In the case of pressurized aerosol, dosage unit can be determined by providing a valve to deliver a measured amount.Capsules and cartridges (for example, gelatin) for use in inhaler or insufflator can be formulated, which contain a powder mix of the compound and suitable powder base, such as lactose or starch.

[0165] Pulmonary delivery of the compound (or salt thereof) is also contemplated. The compound is delivered to the lungs of a mammal during inhalation, crosses the lung epithelial lining, and enters the bloodstream. Other reports on inhaled molecules include: Adjei & Garren, Pulmonary delivery of peptide drugs: Effect of particle size on bioavailability of leuprolide acetate in healthy male volunteers, J Pharmaceutical Research 7: 565-569 (1990); Adjei et al., Bioavailability of leuprolide following intratracheal administration to beagle dogs, International J Pharmaceutics 63:135-144 (1990); Braquet et al., Effect of endothelin-1 on blood pressure and bronchopulmonary system of the guinea pig, J Cardiovascular Pharmacology 13(suppl. 5):143-146 (1989); Hubbard et al., Annals of Internal Medicine 3: 206-212 (1989) (a1-antitrypsin); Smith et al. al., Pulmonary deposition and clearance of aerosolized alpha-1-proteinase inhibitor administered to dogs and to sheep, J Clinical Investigation 84: 1145-1146 (1989)(a1-proteinase);Oswein et al., Aerosolization of Proteins, Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, 1990 (recombinant hepatocyte growth hormone); Debs et al., Lung-specific delivery of cytokines induces sustained pulmonary and systemic immunomodulation in rats, J Immunology 140: 3482-3488 (1988) (interferon-gamma and tumor necrosis factor alpha) and U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor; incorporated herein by reference). Methods and compositions for pulmonary delivery of drugs for systemic effect are described in U.S. Pat. No. 5,451,569 (specifically incorporated herein by reference for the disclosures therein relating to the methods and compositions).

[0166] Contemplated for use are a wide range of mechanical devices designed for pulmonary delivery of therapeutic agents, including, but not limited to, nebulizers, metered dose inhalers, and dry powder inhalers, all of which are well known to those skilled in the art.

[0167] Nasal delivery of pharmaceutical compositions is also contemplated.Nasal delivery allows the passage of pharmaceutical compositions into the bloodstream immediately after administration of the therapeutic agent to the nose, without the need for lung deposition of the therapeutic agent.Formulations for nasal delivery include those that contain dextran or cyclodextran.

[0168] When it is desired to deliver the conjugate systemically, the conjugate can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). The formulation for injection can be presented in a unit dosage form (e.g., in ampoules or multi-dose containers) with added preservatives. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous medium, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0169] Pharmaceutical preparations for parenteral administration include aqueous solutions of active compounds in water-soluble form.In addition, suspensions of active compounds can be prepared as suitable oily injection suspensions.Suitable oily solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound, thereby allowing the preparation of highly concentrated solutions.

[0170] Alternatively, the active conjugates can be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

[0171] The conjugates may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0172] In addition to the formulations described above, the conjugate can also be formulated as a depot preparation.Such long-acting preparations can also be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or can be formulated as sparingly soluble derivatives (e.g., sparingly soluble salts).

[0173] The pharmaceutical compositions may also comprise suitable solid or gel phase or excipients, examples of which include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0174] Suitable liquid or solid pharmaceutical preparations are, for example, in the form of aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated on fine gold particles, contained in liposomes, nebulized, aerosolized, pelletized for skin implantation, or dried on sharp objects for skin injury.The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops, or preparations for sustained release of active compounds, in which excipients and additives and / or auxiliary agents (e.g. disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners, or solubilizers) are customarily used, as described above.The pharmaceutical compositions are suitable for use in various drug delivery systems. For a brief overview of drug delivery methods, see Langer, New methods of drug delivery, Science 249(4976): 1527-1533 (1990).

[0175] The conjugate and optional one or more other therapeutic agents may be administered per se (unmodified) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare their pharmaceutically acceptable salts. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Such salts may also be prepared as alkali metal or alkaline earth metal salts, for example, as sodium, potassium, or calcium salts of carboxylic acid groups.

[0176] Suitable buffering agents include acetic acid and salts (1-2 w / v%), citric acid and salts (1-3 w / v%), boric acid and salts (0.5-2.5 w / v%), and phosphoric acid and salts (0.8-2 w / v%). Suitable preservatives include benzalkonium chloride (0.003-0.03 w / v%), chlorobutanol (0.3-0.9 w / v%), parabens (0.01-0.25 w / v%), and thimerosal (0.004-0.02 w / v%).

[0177] A pharmaceutical composition comprises an effective amount of the compound described herein and, optionally, one or more other therapeutic agents contained in a pharma- ceutically acceptable excipient. The term "pharma-ceutically acceptable excipient" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates. The term "excipient" refers to an organic or inorganic ingredient (natural or synthetic) that is combined with an active ingredient to facilitate application. The components of a pharmaceutical composition may also be mixed with the compound and with each other in such a way that there is no interaction that would substantially impair the desired pharmaceutical efficiency.

[0178] The therapeutic agent, including but not limited to, in particular, a compound, may be provided in a particle. "Particle" as used herein means a nanoparticle or microparticle (or possibly a larger particle) that may comprise in whole or in part a compound or other therapeutic agent described herein. The particle may contain the therapeutic agent in a core surrounded by a coating, including but not limited to an enteric coating. The therapeutic agent may also be dispersed throughout the particle. The therapeutic agent may also be adsorbed to the particle. The particle may be of any order of release kinetics, such as zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof. The particle may include, in addition to the therapeutic agent, any of the materials routinely used in the pharmaceutical and medical fields, such as, but not limited to, erodible, non-erodible, biodegradable, or non-biodegradable materials, or combinations thereof. The particle may be a microcapsule that contains the compound in solution or in a semi-solid state. The particle may be of virtually any shape.

[0179] Both non-biodegradable and biodegradable polymeric materials may be used in the manufacture of particles for delivering therapeutic agents. Such polymers may be natural or synthetic. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include the biodegradable hydrogels described in Sawhney et al., Bioerodible hydrogels based on photopolymerized poly(ethylene glycol)-co-poly(.alpha.-hydroxy acid) diacrylate macromers, Macromolecules 26(4): 581-587 (1993), the teachings of which are specifically incorporated herein by reference. These include: polyhyaluronic acid, casein, gelatin, glutin, polyanhydrase, polyacrylic acid, alginate, chitosan, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0180] The conjugate may be included in a controlled release system. The term "controlled release" is intended to refer to any drug-containing formulation in which the method and profile of drug release from the formulation is controlled. It refers to both immediate release formulations and non-immediate release formulations, including but not limited to sustained release formulations and delayed release formulations. The term "sustained release" (also called "extended release") is used in the conventional sense to refer to a drug formulation that gradually releases drug over an extended period of time and can provide substantially constant blood levels of drug over an extended period of time. The term "delayed release" is used in the conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of drug therefrom. "Delayed release" may or may not involve gradual release of drug over an extended period of time, and therefore may or may not be "sustained release".

[0181] The use of long-term sustained release implants may be particularly suitable for treating chronic conditions. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days and up to 30-60 days. Long-term sustained release implants are known to those skilled in the art and include some of the release systems described above.

[0182] A specific definition The term "about," as used herein, allows for a certain degree of variability in values ​​or ranges (e.g., within 10%, within 5%, or within 1% of a stated value or within the limits of a stated range).

[0183] Values ​​expressed in range format should be interpreted in a flexible manner to include not only the numerical values ​​explicitly recited as limits of the range, but also all individual numerical values ​​or subranges within the range, as if each numerical value and subrange were explicitly recited. For example, the range "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also the individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y" unless otherwise indicated. Similarly, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z" unless otherwise indicated.

[0184] In this document, the terms "a," "an," or "the" are used to include one or more, unless the context clearly indicates otherwise. The term "or" is used to refer to a non-exclusive "or," unless otherwise indicated. In addition, it should be understood that phraseology or terminology employed herein, unless otherwise defined, is for purposes of description only and not for purposes of limitation.

[0185] An "alkyl group" is a group having 1 to 10 carbon atoms (C1 to C 10A is a saturated, partially saturated, or unsaturated, linear or branched acyclic hydrocarbon having 1 to 8 carbons (C1-C8 alkyl), 1 to 6 (C1-C6 alkyl), 1 to 4 (C1-C4 alkyl), 1 to 3 (C1-C3 alkyl), or 2 to 6 (C2-C6 alkyl) carbon atoms. In some embodiments, the alkyl group has a monovalency. Examples of alkyl groups having a monovalency include -CH3, -CH2CH3, and the like. Monovalent alkyls may be found in substituents, for example, in the chain of the linker L. In some embodiments, the alkyl group has a divalent nature, for example, when found in the chain of the linker L. Examples of alkyl groups having a divalent nature include, but are not limited to, -CH2-, -CH2CH2-, and the like. In some embodiments, the alkyl group is a saturated alkyl group. In some embodiments, the alkyl group is an unsaturated alkyl group, also referred to as an alkenyl or alkynyl group.

[0186] The term "heteroalkyl," alone or in combination with another term, means, unless otherwise specified, a stable linear or branched chain or combination thereof consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH2=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two heteroatoms may be consecutive (e.g., -CH2-NH-OCH3).

[0187] Any use of section headings and subheadings is for ease of reference only and is not intended to limit any disclosure made in a section to only that section; rather, any disclosure made under a section heading or subheading is intended to constitute disclosure under all other section headings or subheadings.

[0188] Various modifications and alterations of the described compositions, methods, and uses of the techniques will be apparent to those skilled in the art without departing from the scope and spirit of the described techniques. Although the techniques have been described in connection with specific exemplary embodiments, the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.

[0189] The terms and expressions utilized are used as terms of description and not of limitation. In this regard, where a particular term is defined and described or discussed elsewhere, the definition and all descriptions and discussions are intended to be attributable to such terms. Furthermore, the use of such terms and expressions is not intended to exclude equivalents of any illustrated and described features or portions thereof.

[0190] Additionally, all publications and patents mentioned herein are incorporated by reference in their entirety for all purposes. In the event of inconsistencies in usage between this document and a document incorporated by reference, the usage in the incorporated reference should be considered as supplementary to the usage in this document, and in the event of irreconcilable discrepancies, the usage in this document will take precedence.

[0191] Embodiment The following embodiments, presented as items, are not intended to limit the invention of the present disclosure, but are intended to further illustrate the invention of the present disclosure.

[0192] Item A. Formula: TL-LH n or a pharma- ceutically acceptable salt thereof, During the ceremony, TLs are targeting ligands for target proteins on the surface of viruses, virus-infected cells, cancer cells, immune cells, or fibroblasts, L is a linker, H is a hapten, n is an integer from 2 to 3; Optionally, at least two of H are each capable of binding to a different antibody when contacted with said different antibody; The conjugate, or a pharma- ceutically acceptable salt thereof.

[0193] Item B. The conjugate of item A, wherein at least two of H are each bound to an antibody.

[0194] Item C. The conjugate of items A or B, wherein each H is bound to a different antibody.

[0195] Item D. The conjugate of any one of items A-C, wherein each H is independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof.

[0196] The conjugate according to item A, wherein item En is 2.

[0197] The conjugate according to claim A, wherein Fn is 3.

[0198] Item G. The conjugate of any one of items A-C, E, or F, wherein each H is independently selected from a rhamnose fragment, an α-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin, or from a viral antigen selected from diphtheria, varicella zoster virus, human papilloma virus, influenza virus, SARS-COV-2, yellow fever, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis G, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox, monkeypox, poliovirus, dengue virus, and measles virus.

[0199] The conjugate according to any one of items A to C, wherein item Hn is 2, the first H is a DNP fragment and the second H is a rhamnose fragment.

[0200] Item I. The conjugate of item A, wherein at least one H is an influenza virus antigen selected from hemagglutinin and neuraminidase.

[0201] Item J. The conjugate of item A, wherein at least one H is a hepatitis antigen selected from L-HBsAg, S-HBsAg, M-HBsAg, and preS.

[0202] Item K. The conjugate of item A, wherein at least one H is gp120 or gp160.

[0203] Item L. The conjugate of item A, wherein at least one H is a glycoprotein.

[0204] Item M. The conjugate of any one of items A-C, E, F, or I-L, wherein the target protein is a viral envelope protein or a viral envelope protein on the surface of a virally infected cell.

[0205] Item N. The conjugate of any one of items A-C, E, F, or I-L, wherein the target protein is influenza neuraminidase or influenza hemagglutinin.

[0206] Item O. The conjugate of any one of items A-C, E, F, or I-L, wherein the target protein is respiratory syncytial virus fusion protein F.

[0207] Item P. The conjugate of items A-C, E, F, or I-L, wherein the target protein is a coronavirus spike protein.

[0208] Item Q. The conjugate of items A-C, E, F, or I-L, wherein the target protein is Hepatitis B virus surface antigen or HBV core antigen.

[0209] Item R. The conjugate of items A-C, E, F, or I-L, wherein the target protein is a cell surface receptor on a cancer cell.

[0210] Item S. The conjugate of item A, wherein the target protein is a folate receptor.

[0211] Item T. The conjugate of item S, wherein the target protein is folate receptor alpha or folate receptor beta.

[0212] Item U. The conjugate of any one of items A-C, E, F, or I-L, wherein the target protein is prostate specific membrane antigen.

[0213] Item V. The conjugate of any one of items A to C, E, F, or I to L, wherein the target protein is carbonic anhydrase 9.

[0214] Item W. The conjugate of any one of items A-C, E, F, or I-L, wherein the target protein is a luteinizing hormone releasing hormone receptor.

[0215] Item X. The conjugate of any one of items A to C, E, F, or I to L, wherein the target protein is a neurokinin 1 receptor.

[0216] Item Y. The conjugate of any one of items A-C, E, F, or I-L, wherein the target protein is a cell surface receptor on tumor associated macrophages.

[0217] Item Z. The conjugate of any one of items A-C, E, F, or I-L, wherein the target protein is a cell surface receptor on a myeloid-derived suppressor cell.

[0218] Item AA. The conjugate of any one of items AC, E, F, or I-L, wherein the target protein is a cell surface receptor on a cancer-associated fibroblast.

[0219] Paragraph BB. The conjugate of any one of paragraphs AC, E, F, or I-L, wherein the target protein is a fibroblast activation protein.

[0220] Item CC. The conjugate of any one of items AC, E, F, or IL, wherein the targeting ligand is a neuraminidase inhibitor.

[0221] Item DD. The conjugate of any one of items AC, E, F, or IL, wherein the targeting ligand is an oseltamivir fragment, a zanamivir fragment, a peramivir fragment, or a laninamivir fragment.

[0222] Item EE. The conjugate of item A, wherein the targeting ligand is a zanamivir fragment.

[0223] Item FF. The conjugate of any one of items AC, E, F, IL, S, or T, wherein the targeting ligand is a folic acid fragment or an analog thereof.

[0224] Item GG. The conjugate of any one of items AC, E, F, IL, S, or T, wherein the targeting ligand is 5-methyltetrahydrofolate.

[0225] Item HH.L is (-CH2CH2-O-) n (wherein n is an integer from 1 to 32, inclusive), a peptide, a peptidoglycan, or a combination of two or more of the foregoing.

[0226] The conjugate of any one of paragraphs A-C, E, F, I-L, S, T, or EE, wherein paragraph II.L is a branched linker, and at least two of the haptens are attached to different branches of the linker, and the different branches optionally extend from different atoms of the linker.

[0227] Item JJ. The conjugate of item A, wherein the targeting ligand is a folate fragment or a derivative thereof, at least a first H comprises a rhamnose fragment, and at least a second H comprises a dinitrophenyl fragment.

[0228] Item KK. The conjugate of any one of items A-JJ formulated as a prodrug.

[0229] Item LL.Formula:

[0230] [ka] or a pharma- ceutically acceptable salt thereof, During the ceremony, TLs are targeting ligands for target proteins on the surface of viruses, virus-infected cells, cancer cells, immune cells, or fibroblasts, L a , L b , and L care each a linker, which may be the same or different, C is a carbon atom, R4 is selected from hydrogen, a C1-C5 alkyl group, a C1-C5 alkenyl group, or a C1-C5 alkynyl group; H1 and H2 are each a hapten; Optionally, H1 and H2 can each bind to a different antibody. The conjugate, or a pharma- ceutically acceptable salt thereof.

[0231] Item MM.Formula:

[0232] [ka] or a pharma- ceutically acceptable salt thereof, During the ceremony, TLs are targeting ligands for target proteins on the surface of viruses, virus-infected cells, cancer cells, immune cells, or fibroblasts, L a , L b , L c , and L d are each a linker, which may be the same or different, C is a carbon atom, H1, H2, and H3 are each a hapten; Optionally, each H1, H2, and H3 can each bind to a different antibody. The conjugate, or a pharma- ceutically acceptable salt thereof.

[0233] In paragraphs NN.H1 and H2, respectively, a conjugate according to paragraph MM to which an antibody is bound.

[0234] Items OO.H1, H2, and H3 each include a conjugate according to item MM to which an antibody is attached.

[0235] The conjugate according to paragraphs LL or NN, wherein each of paragraphs PP.H1 and H2 is independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof.

[0236] The conjugate according to paragraphs MM or PP, wherein H1, H2, and H3 are each independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof.

[0237] A conjugate according to item LL or NN, wherein item RR.H1 or H2 is each independently selected from a rhamnose fragment, an alpha-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin, or selected from a viral antigen selected from diphtheria, varicella zoster virus, human papilloma virus, influenza virus, SARS-COV-2, yellow fever, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis G, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox, monkeypox, poliovirus, dengue virus, and measles virus.

[0238] The conjugate according to paragraphs LL or OO, wherein H1, H2, or H3 are each independently selected from a rhamnose fragment, an alpha-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin, or from a viral antigen selected from diphtheria, varicella zoster virus, human papilloma virus, influenza virus, SARS-COV-2, yellow fever, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis G, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox, monkeypox, poliovirus, dengue virus, and measles virus.

[0239] A conjugate according to paragraphs LL or OO, wherein paragraph TT.H1 is a DNP fragment and H2 is a rhamnose fragment.

[0240] The conjugate according to paragraph UU or MM, wherein at least one hapten is an influenza virus antigen selected from hemagglutinin and neuraminidase.

[0241] Item VV. The conjugate of items LL or MM, wherein at least one hapten is a hepatitis antigen selected from L-HBsAg, S-HBsAg, M-HBsAg, and preS.

[0242] Item WW. The conjugate according to item LL or MM, wherein at least one hapten is gp120 or gp160.

[0243] Item XX. The conjugate according to item LL or MM, wherein at least one hapten is a glycoprotein.

[0244] Item YY. The conjugate of any one of items LL-OO, wherein the target protein is a viral envelope protein or a viral envelope protein on the surface of a virally infected cell.

[0245] Item ZZ. The conjugate of any one of items LL-OO, wherein the target protein is influenza neuraminidase or influenza hemagglutinin.

[0246] Item AAA. The conjugate of any one of items LL-OO, wherein the target protein is respiratory syncytial virus fusion protein F.

[0247] Item BBB. The conjugate of any one of items LL-OO, wherein the target protein is a coronavirus spike protein.

[0248] Item CCC. The conjugate of any one of items LL-OO, wherein the target protein is Hepatitis B virus surface antigen or HBV core antigen.

[0249] Item DDD. The conjugate of any one of items LL-OO, wherein the target protein is a cell surface receptor on a cancer cell.

[0250] Item EEE. The conjugate of any one of items LL-OO, wherein the target protein is a folate receptor.

[0251] Item FFF. The conjugate of any one of items LL-OO, wherein the target protein is folate receptor alpha or folate receptor beta.

[0252] Item GGG. The conjugate of any one of items LL-OO, wherein the target protein is prostate specific membrane antigen.

[0253] Item HHH. The conjugate of any one of items LL-OO, wherein the target protein is carbonic anhydrase 9.

[0254] Item III. The conjugate according to any one of items LL-OO, wherein the target protein is a luteinizing hormone releasing hormone receptor.

[0255] Item JJJ. The conjugate of any one of items LL-OO, wherein the target protein is a neurokinin 1 receptor.

[0256] Item KKK. The conjugate of any one of items LL-OO, wherein the target protein is a cell surface receptor on tumor-associated macrophages.

[0257] Item LLL. The conjugate of any one of items LL-OO, wherein the target protein is a cell surface receptor on a myeloid-derived suppressor cell.

[0258] Item MMM. The conjugate of any one of items LL-OO, wherein the target protein is a cell surface receptor on cancer-associated fibroblasts.

[0259] Item NNN. The conjugate of any one of items LL-OO, wherein the target protein is a fibroblast activation protein.

[0260] Item OOO. The conjugate of any one of items LL-OO, wherein the targeting ligand is a neuraminidase inhibitor.

[0261] Item PPP. The conjugate according to any one of items LL to OO, wherein the targeting ligand is an oseltamivir fragment, a zanamivir fragment, a peramivir fragment, or a laninamivir fragment.

[0262] Item QQQ. The conjugate of any one of items LL-OO, wherein the targeting ligand is a zanamivir fragment.

[0263] Item RRR. The conjugate of any one of items LL-OO, wherein the targeting ligand is a folic acid fragment or an analog thereof.

[0264] Item SSS. The conjugate of any one of items LL-OO, wherein the targeting ligand is 5-methyltetrahydrofolate.

[0265] Item TTT.L a , L b , L c , and L d At least one of each independently represents (-CH2CH2-O-) n wherein n is an integer from 1 to 32, inclusive; an alkyl group; a peptide; a peptidoglycan; or a combination of two or more of the foregoing.

[0266] Item UUU.L a , L b , and L c At least one of each independently represents (-CH2CH2-O-) n (wherein n is an integer from 1 to 32, inclusive), an alkyl group, a peptide, a peptidoglycan, or a combination of two or more of the foregoing.

[0267] A conjugate according to paragraphs TTT or UUU, wherein paragraph VVV.n is an integer from 1 to 16, inclusive.

[0268] Item WWW.L a , L b , and L c , and L d The conjugate of paragraphs MM or OO, wherein at least one of comprises a peptide fragment or a peptidoglycan fragment.

[0269] Item XXX.L a , L b , and L c The conjugate of paragraphs MM or NN, wherein at least one of comprises a peptide fragment or a peptidoglycan fragment.

[0270] Item ZZZ.L a , L b , and L c , L d are respectively independent of each other, C2 to C 18 A conjugate according to paragraphs MM or OO, comprising an alkyl group.

[0271] Item AAAA.L a , L b , and L c are respectively independent of each other, C2 to C 18 A conjugate according to paragraphs LL or NN which comprises an alkyl group.

[0272] Item BBBB.Formula:

[0273] [ka] or a pharma- ceutically acceptable salt thereof.

[0274] Item CCCC.Formula:

[0275] [ka] or a pharma- ceutically acceptable salt thereof.

[0276] The conjugate according to paragraphs AAAA or BBBB, wherein one or more of the --OH groups are independently replaced with a thiol, a phosphate, or a phosphanate ester.

[0277] Item EEEE. The conjugate according to item AAAA or BBBB, wherein one or more of the -OH groups are replaced with -OC(=O)R, where R is an alkyl group.

[0278] Item FFFF. The conjugate according to item AAAA or BBBB, wherein one or more of the -OH groups are replaced with -OC(=O)R, where R is a C1-C6 alkyl group.

[0279] The conjugate according to item GGGG., wherein the amine (-NH2) group is replaced with -OC(=O)R2, wherein R2 is an alkyl group.

[0280] Item HHHH. The conjugate according to item AAAA or BBBB, wherein the amine (-NH2) group is replaced with -OC(=O)R2, where R2 is a C1-C6 alkyl group.

[0281] Item III. The conjugate according to item AAAA or BBBB, wherein the carboxyl (-COOH) group is replaced with -OC(=O)R3, where R3 is an alkyl group.

[0282] Item JJJJ. The conjugate according to item AAAA or BBBB, wherein the carboxyl (-COOH) group is replaced with -OC(=O)R3, wherein R3 is a C1-C6 alkyl group.

[0283] Item KKKK.Formula:

[0284] [ka] or a pharma- ceutically acceptable salt thereof, wherein L1, L2, and L3 are linkers.

[0285] One or more of the items LLLL.L1, L2, and L3 is (-CH2CH2-O-) n (wherein n is an integer from 1 to 16, inclusive).

[0286] Items MMMM.L1, L2, and L3 are each independently C2 to C 18 A conjugate according to paragraphs JJJJ or KKKK which comprises an alkyl group, a peptide fragment, or a peptidoglycan fragment.

[0287] Item MMMM.Formula:

[0288] [ka] or a pharma- ceutically acceptable salt thereof.

[0289] Item NNNN. The conjugate of any one of items AAAA-MMMM, which is further conjugated in vivo to one or more antibodies.

[0290] Item OOOO. A pharmaceutical composition comprising a conjugate according to any one of items A to MMMM and a pharma- ceutically acceptable excipient.

[0291] Item PPPP. A method for treating a viral infection in a subject, comprising administering to the subject an effective amount of a conjugate described in any one of items A to MMMM or a pharmaceutical composition described in item OOOO.

[0292] The method of claim PPPP, further comprising administering to the subject an autoantibody or an allogeneic immunoglobulin G (IgG) antibody.

[0293] Item RRRR. The method according to Item PPPP, wherein the viral infection is influenza.

[0294] Item SSSS. The method of any one of Items PPPP to RRRR, wherein the conjugate or pharmaceutical composition is administered orally.

[0295] The method according to item PPPP, wherein the conjugate or the pharmaceutical composition is administered once a day.

[0296] Item UUUU. The method according to Item PPPP, wherein the conjugate or pharmaceutical composition is administered multiple times daily.

[0297] Item VVVV. The method according to Item PPPP, wherein the conjugate or the pharmaceutical composition is administered twice a day.

[0298] Item WWWW. A method for treating cancer in a subject, comprising administering to the subject an effective amount of a conjugate described in any one of Items A to MMMM or a pharmaceutical composition described in Item OOOO.

[0299] Item XXXX. The method of Item WWWW, further comprising administering to the subject an autoantibody or an allogeneic IgG antibody.

[0300] Item YYYY. The method according to Item WWWW, wherein the cancer is a hot cancer.

[0301] The method of claim WWWW, wherein the cancer is renal cancer, lung cancer, or colorectal cancer.

[0302] Item AAAAA. The method of any one of Items PPPP-ZZZZ, wherein the conjugate or the pharmaceutical composition is administered orally or intravenously.

[0303] Item BBBBB. The method of any one of Items PPPP-ZZZZ, wherein the conjugate or pharmaceutical composition is administered once daily.

[0304] Item CCCCC. The method of any one of Items PPPP-ZZZZ, further comprising administering to the subject a second therapeutic agent, wherein the second therapeutic agent comprises a chemotherapeutic agent, sunitinib, a PD-1 inhibitor, or a PDL-1 inhibitor.

[0305] Item DDDDD. A method for activating an immune response in a subject, comprising administering to the subject an effective amount of a conjugate described in any one of items A to MMMM or a pharmaceutical composition described in item OOOO.

[0306] Item EEEEE. The method of Item DDDDD, wherein the immune response is an innate immune response.

[0307] The method of claim DDDDD, wherein the immune response is activated within a target area of ​​the subject, the target area being the tumor microenvironment or the location of a site of viral replication.

[0308] Item GGGGG. The method of any one of items DDDDD-FFFFF, further comprising administering to the subject an autoantibody or an allogeneic IgG antibody.

[0309] Item HHHHH. The method according to item FFFFF, wherein administration of an effective amount of said conjugate or said pharmaceutical composition induces reprogramming of M2 type macrophages to M1 type macrophages in said target area.

[0310] Item IIIII. The conjugate of item A, wherein at least two of H are each capable of binding to a different antibody when contacted with an antibody in vivo.

[0311] Item JJJJJ. The conjugate of item A, wherein two of H are each capable of binding to a different antibody when contacted with the antibody in vitro.

[0312] [Table 1-1]

[0313] [Table 1-2] EXAMPLES

[0314] The following examples serve to illustrate the present disclosure and are not intended to limit the scope of the claims in any way.

[0315] [Example 1] Synthesis of Zan-PEG6-DNP-Rhamnose (Zan-Dual Hapten) Drug Conjugate (Compound 24)

[0316] [ka]

[0317] [ka]

[0318] Reagents and conditions: a) PPh3, THF, and HO; b) N,N'-di-Boc-1H-pyrazole-1-carboxamidine, tetrahydrofuran (THF), and triethylamine (TEA); c) NaOMe, MeOH; d) 2,2-dimethoxypropane, dry acetone, p-TsOH; e) 4-nitrophenyl chloroformate, 4-dimethylaminopyridine (DMAP), and pyridine at room temperature; f) N3-PEG6-NH2, N,N-diisopropylethylamine (DIPEA), and THF; g) 1M NaOH(aq), THF; h) trifluoroacetic acid (TFA) at room temperature.

[0319] Synthesis scheme of compound 12:

[0320] [ka]

[0321] Synthesis scheme of compound 16:

[0322] [ka]

[0323] Synthesis scheme of zan-DNP-rhamnose (compound 24):

[0324] [ka]

[0325] [ka]

[0326] Ac is acetate.

[0327] Synthesis of compound 2: Zanamivir derivative 1 was prepared from sialic acid according to the literature methods reported in: Chandler et al., Synthesis of the potent influenza neuraminidase inhibitor 4-guanidino Neu5Ac2en. X-Ray molecular structure of 5-acetamido-4-amino-2, 6-anhydro-3, 4, 5-trideoxy-Derythro-L-gluco-nononic acid, J Chem Society, Perkin Transactions 1: 1173-1180 (1995); Shidmoossavee et al., Chemical insight into the emergence of influenza virus strains that are resistant to Relenza, J Am Chem Soc 135: 13254-13257 (2013); and Ying & Gervay-Hague, One-Bead-One-Inhibitor-One-Substrate Screening of Neuraminidase Activity, ChemBioChem 6: 1857-1865 (2005). To a solution of zanamivir intermediate (1) (5 g, 11 mmol) in THF (40 mL) was added triphenylphosphine (3.67 g, 14 mmol, 1.27 equiv.) and the resulting solution was stirred at room temperature for 12 h. Water (10 mL) was then added and the solution was stirred at room temperature for an additional 24 h. The reaction solution was then concentrated and the crude product was purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel column, 0-100% EtOAc in hexanes) to give 2 as a yellow powder. The product 2 was isolated in 2.89 g, 61% yield.

[0328] Synthesis of compound 3: To a solution of compound 2 (2.74 g, 6.37 mmol) and N,N'-bis(tertbutoxycarbonyl)-1H-pyrazole-1-carboxamidine (2.57 g, 8.28 mmol, 1.30 equiv.) dissolved in THF (20 mL) was added triethylamine (1.5 mL). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel column, 0-100% EtOAc in hexanes) to give compound 3 as a white solid (4.14 g, 97%).

[0329] Synthesis of compound 5: To a stirred solution of compound 3 (4.225 g, 6.281 mmol) in anhydrous methanol (70 mL) was added NaOMe (2.9 mL, 0.5 M, 1.414 mmol). The reaction mixture was then stirred for 1 h. Dowex 50XW8 (H + ) resin was added to neutralize the reaction mixture, filtered and concentrated to give compound 4, which was used in the next step without further purification.

[0330] To compound 4 in dry acetone (70 mL) was added 2,2-dimethoxypropane (7.7 mL, 6.54 g, 62.81 mmol, 10 equiv.), followed by p-toluenesulfonic acid (120 mg, 0.628 mmol, 0.1 equiv.), and the resulting mixture was stirred at room temperature overnight. The reaction mixture was then concentrated and purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel column, 0-100% EtOAc in hexanes) to give compound 5 as a white solid, isolated product 2.4 g, and 65% yield.

[0331] Synthesis of compound 6: To a solution of compound 5 (1.46 g, 2.49 mmol) in pyridine (30 mL), 4-dimethylaminopyridine (2.13 g, 17.43 mmol) and 4-nitrophenyl chloroformate (3.51 g, 17.43 mmol) were added. The reaction mixture was stirred at room temperature overnight, then concentrated and purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel column, 0-100% EtOAc in hexanes) to give compound 6 as a white solid (1.62 g, 87%).

[0332] Synthesis of compound 7: To a solution of activated zanamivir, compound 6 (0.1 g, 0.13 mmol) in THF (2.7 mL), N3-PEG6-NH2 (0.05 g, 0.14 mmol, 1.05 equiv.) was added at room temperature under argon, followed by DIPEA (0.12 mL, 0.67 mmol, 5.0 equiv.) and stirred overnight. The progress of the reaction was monitored by thin layer chromatography (TLC) and LC / MS.

[0333] After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The crude product was purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel column, 0-10% MeOH in DCM) to give compound 7 as a gummy solid (0.12 g, 93%) (see FIG. 2A).

[0334] Synthesis of compound 9: Compound 7 (75 mg, 0.078 mmol) was dissolved in THF:MeOH (6:1, 1.5 mL) and treated dropwise with 1M NaOH (100 μL). The reaction mixture was stirred at room temperature for 1 h, at which point LC-MS analysis revealed that the deprotection of the methyl ester was complete. The reaction mixture was chromatographed over 100 mL of Dowex® 50WX8 (H + The mixture was neutralized by addition of 100% hexanes (100% ethanol), filtered, and concentrated under reduced pressure. The crude intermediate product 8 was used directly in the next step without further purification (see FIG. 2B).

[0335] To the intermediate crude compound 8 was added TFA (0.5 mL). The reaction mixture was stirred at room temperature for 1 h and LC-MS showed the reaction was complete. TFA was removed by rotary evaporation under reduced pressure and the crude product was placed under vacuum to give compound 9. The overall yield over the two steps was 91% (see FIG. 2C).

[0336] Synthesis of DNP-PEG1-CO2H (compound 12): To a solution of 1-chloro-2,4-dinitrobenzene, compound 10 (0.5 g, 2.47 mmol), and 3-(2-aminoethoxy)propanoic acid, compound 11 (0.33 g, 2.47 mmol) dissolved in EtOH (25 mL), TEA (1.38 mL, 9.87 mmol) was added. The reaction mixture was heated to 55° C. for 16 h and the progress of the reaction was monitored by LC-MS. After completion of the reaction (as confirmed by the disappearance of one of the starting materials, i.e., dinitrobenzene), the reaction mixture was cooled and concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel column, 0-20% methanol in DCM) to give compound 12 as a yellow solid (90% yield). LC-MS [M+H]+=299.98. (See Figure 2D).

[0337] Synthesis of compound 14: α-L-rhamnose monohydrate (compound 13, 1.0 g, 5.49 mmol) was dissolved in 9.2 mL of anhydrous pyridine. The solution was stirred in an ice bath and purged with nitrogen before adding acetic anhydride (4.15 mL, 43.92 mmol, 8.0 equiv.) dropwise. The reaction mixture was allowed to slowly reach room temperature and monitored by TLC (hexane / EtOAc, 65:35) and LC-MS, which showed complete consumption of α-L-rhamnose monohydrate after 20 h of reaction under inert atmosphere. The reaction mixture was poured into ethyl acetate and extracted twice with 1.0 M HCl. The organic layer was washed with saturated sodium carbonate solution, water, and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The resulting crude oily product (compound 14) (98% yield) was used in the next step.

[0338] Synthesis of compound 16: 1,2,3,4-tetra-O-acetyl-α-L-rhamnose (compound 14, 0.50 g, 1.50 mmol) was dissolved in DCM (7.5 mL) and then amino-PEG4-OH (compound 15, 0.35 g, 1.81 mmol, 1.2 equiv.) was added under inert atmosphere. The reaction flask was placed in an ice bath and boron trifluoride diethyl etherate (0.56 mL, 4.51 mmol, 3.0 equiv.) was added dropwise over 30 min. The reaction mixture was stirred in an ice bath for 2 h after which the reaction reached room temperature. TLC (hexane / EtOAc, 30:70, Rf=0.30) and LC-MS monitoring showed complete consumption of 1,2,3,4-tetra-O-acetyl-α-L-rhamnose after 16 h of reaction. The reaction mixture was poured into ice water and extracted with DCM. The combined organic layers were washed twice with saturated sodium bicarbonate solution, water, and brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The crude material obtained was purified by silica gel column chromatography (hexane / EtOAc, 20:80) to isolate the product (compound 16) (yield 90%).

[0339] Synthesis of compound 18: To a solution of 3-(2-((2,4-dinitrophenyl)amino)ethoxy)propanoic acid (compound 12, 0.1 g, 0.33 mmol) in dimethylsulfoxide (2 mL) under argon atmosphere, 1-[bis(dimethylamino)-methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, HATU (0.11 g, 0.28 mmol, 0.85 equiv.), followed by DIPEA (0.29 mL, 1.67 mmol, 5.0 equiv.) was added and stirred at room temperature for 10 min. To this reaction mixture, Fmoc-Lys-OH.HCl (compound 17, 0.11 g, 0.27 mmol, 0.8 equiv.) was added and stirred at room temperature for 2-3 h. The progress of the reaction was monitored by LC-MS. After the completion of the reaction was confirmed by LC-MS, the reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The resulting crude material was purified by silica gel column chromatography (hexane / EtOAc, 20:80) to isolate the product (compound 18) (70% yield) (see FIG. 2E).

[0340] Synthesis of compound 19: To a solution of acid (compound 18, 0.06 g, 0.09 mmol) and Rham(OAc)3-PEG4-NH2 (compound 16, 0.04 g, 0.09 mmol, 1.0 equiv.) in dimesyl sulfoxide (1.5 mL), benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, PyBOP (0.05 g, 0.10 mmol, 1.1 equiv.) was added at room temperature under argon atmosphere, followed by DIPEA (0.081 mL, 0.46 mmol, 5.0 equiv.). The progress of the reaction was monitored by LC / MS.

[0341] After the reaction was complete by LC-MS, the reaction mixture was quenched by the addition of water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude material obtained was purified by silica gel column chromatography (0-10% MeOH in DCM) to isolate the product (compound 19) (yield 93%).

[0342] Deprotection of -Fmoc group (compound 20): To compound 19 (0.01 g, 0.01 mmol) in dry DCM (0.2 ml) was added DEA (100 uL) at room temperature under argon. The solution was stirred at room temperature for 1 h until the reaction was complete as demonstrated by LC-MS. DEA was removed by rotary evaporation under reduced pressure and the crude product was precipitated in diethyl ether to give compound 20 as a yellow solid in quantitative yield. LC-MS [M+H]+=876.1.

[0343] Synthesis of compound 22: To a solution of DBCO-NHS (compound 21, 0.012 g, 0.029 mmol, 1.05 eq.) and compound 20 (0.024 g, 0.027 mmol, 1.0 eq.) dissolved in DCM:DMSO (10:1, 0.6 mL) under argon, N,N-DIPEA (0.048 μl, 0.27 mmol, 10 eq.) was added dropwise. The reaction mixture was stirred at room temperature for 2-3 h and the progress of the reaction was monitored by LC-MS. After completion of the reaction, the solvent was evaporated using a rotary evaporator under reduced pressure and the crude material was purified by silica gel column chromatography on a Teledyne CombiFlash Rf+ Lumen (0-10% methanol in DCM) to give compound 22 as a yellowish solid (93% yield). LC-MS [M+H]+=1163.2. (See Figure 2F).

[0344] Hydrolysis of OAc group (compound 23): Compound 22 (0.025 g, 0.022 mmol) was dissolved in anhydrous MeOH (1 mL) and treated dropwise with 0.5 M NaOH (25 μL). The reaction mixture was stirred at room temperature for 1 h, at which point LC-MS analysis revealed the formation of the product. After completion of the reaction, the reaction mixture was neutralized by adding Dowex® 50WX8 (H+) resin, filtered, and concentrated under reduced pressure. The yellowish crude product (compound 23) was used directly in the next step without further purification. LCMS [M+H]+=1037.3. (See FIG. 2G).

[0345] Synthesis of zan-PEG6-DNP-rhamnose (compound 24): To a solution of zanamivir-PEG6-azide (compound 9) (2.4 mg, 0.003 mmol) and compound 23 (3.5 mg, 0.003 mmol, 1.0 equiv) in dry DCM:DMSO (10:1, 0.2 mL) was added DIPEA (6 μl, 0.03 mmol, 10 equiv) dropwise under argon. The reaction mixture was stirred at room temperature (RT) for 2 h and the progress of the reaction was monitored by LC-MS. After completion of the reaction, the solvent was removed by rotary evaporation under reduced pressure and the crude product was purified by preparative high performance liquid chromatography (HPLC) on a C18 column (5-95% acetonitrile in aqueous 20 mM NH4OAc, pH 7 buffer, 60 min, flow rate 7 mL / min) to give Zan-PEG6-DNP-rhamnose (compound 24) as a yellow powder (yield 50%). LC-MS [M+H]+=1745.9. (See Figure 2H).

[0346] [Example 2] Comparison of compound 24 with zan-Fc-WT and zan-Fc-DLE Six to eight week old female BALB / c mice (n=5 / group) were administered 100 LD 50Mice were infected with influenza virus A / Puerto Rico / 8 / 1934 (H1N1) (catalog number NR-348, BEI Resources, NIAID, NIH) from the University of Illinois. Mice (except for the phosphate-buffered saline (PBS) alone group) were injected intraperitoneally with 8 g / kg human intravenous immunoglobulin (IVIg) at 24 hours post-infection (hpi) to achieve humanized titers of anti-DNP and anti-rhamnose antibodies at the time of test article administration. Mice were treated with test articles at 48 hpi. Zan-DNP-rhamnose was administered as a single intranasal dose of 1.5 μmol / kg. zan-Fc-WT and zan-Fc-DLE were administered as a single intravenous dose of 10 μg / mouse (synthesis detailed below). Two control groups of mice received PBS as a placebo.

[0347] To assess efficacy, mice were monitored by daily weighing for 14 days post-infection and were considered dead when they lost 25% of their body weight or were diagnosed as moribund. The results of this experiment are shown in Figures 3 and 4.

[0348] Figure 3 shows the 100LD 50 Graph of days post infection versus % survival for mice (n=5 / group) infected with influenza A H1N1 / PR8, clearance of anti-influenza antibodies, intraperitoneal administration of IVIg (GAMUNEX®-C) at 24 hpi, and administration of the conjugate at 48 hpi.

[0349] FIG. 4 is a graph of days post-infection versus body weight (%) for mice treated as described in FIG.

[0350] [ka]

[0351] [ka]

[0352] Zanamivir-azide, compound 4″, was prepared from sialic acid according to previously reported literature methods (Chandler et al., Synthesis of the potent influenza neuraminidase inhibitor 4-guanidino Neu5Ac2en. X-ray molecular structure of 5-acetamido-4-amino-2,6-anhydro-3,4,5-trieoxy-D-erythro-L-gluco-nononic acid, J Chemical Society, Perkin Transactions 1: 1173-1180 (1995); Shidmoossavee et al., Chemical insight into the emergence of influenza virus strains that are resistant to Relenza, J American Chemistry Society 135(36): 13254-13257 (2013); Ying, L. & Gervay-Hague, One-bead-one-inhibitor-one-substrate screening of neuraminidase activity, ChemBioChem 6(10): 1857-1865 (2005).

[0353] Synthesis of compound 5″: To a solution of zanamivir-azide (compound 4″) (5 g, 11 mmol) in THF (40 mL), triphenylphosphine (3.67 g, 14 mmol, 1.27 equiv.) was added and the resulting solution was stirred at room temperature for 12 h. Water (10 mL) was then added and the solution was stirred at room temperature for an additional 24 h. The reaction solution was then concentrated and the crude product was purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel column, 0-100% EtOAc in hexanes) to give compound 5″ as a yellow powder. The product (compound 5″) was isolated in 2.89 g; yield 61.1%.

[0354] Synthesis of compound 6": To a solution of compound 5" (2.74 g, 6.37 mmol) and N,N'-bis(tertbutoxycarbonyl)-1H-pyrazole-1-carboxamidine (2.57 g, 8.28 mmol, 1.30 equiv) dissolved in THF (20 mL) was added triethylamine (1.5 mL). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel column, 0-100% EtOAc in hexanes) to give compound 7" as a white solid (4.14 g, 97%).

[0355] Synthesis of Compound 8": To a stirred solution of compound (compound 6) (4.225 g, 6.281 mmol) in anhydrous methanol (70 mL), NaOMe (2.9 mL, 0.5 M, 1.414 mmol) was added. The reaction mixture was then stirred for 1 h. Dowex 50XW8 (H + ) resin was added to neutralize the reaction mixture, and the mixture was filtered and concentrated to give the lead compound 7″, which was used in the next step without further purification.

[0356] To compound 7″ in dry acetone (70 mL) was added 2,2-dimethoxypropane (7.7 mL, 6.54 g, 62.81 mmol, 10 equiv.) followed by p-toluenesulfonic acid (120 mg, 0.628 mmol, 0.1 equiv.). The resulting mixture was stirred at room temperature overnight. The reaction mixture was then concentrated and purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel column, 0-100% EtOAc in hexanes) to give compound 8″ as a white solid (2.4 g, 65.2%).

[0357] Synthesis of compound 9″: To a solution of compound 8″ (1.46 g, 2.49 mmol) in pyridine (30 mL) was added 4-dimethylaminopyridine (2.13 g, 17.43 mmol) and 4-nitrophenyl chloroformate (3.51 g, 17.43 mmol). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel column, 0-100% EtOAc in hexanes) to give activated zanamivir (compound 9″) as a white solid (1.62 g, 87%).

[0358] Synthesis of compound 10″: To a solution of activated zanamivir (compound 9″) (0.2 g, 0.27 mmol) in THF (12.0 mL) under argon at room temperature, tert-Boc-N-amido-PEG6-amine (0.124 g, 0.29 mmol, 1.1 equiv.) was added followed by DIPEA (0.23 mL, 1.33 mmol, 5.0 equiv.) and stirred for 6-12 h. The progress of the reaction was monitored by TLC and LC / MS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The crude product was purified by flash column chromatography on a Teledyne CombiFlash Rf+ Lumen (silica gel column, 0-20% MeOH in DCM) to give compound 10″ as a gummy solid (234 mg, 85%).

[0359] Synthesis of compound 12": Compound 10" (0.22 g, 0.21 mmol) was dissolved in THF (1.5 mL) and treated dropwise with 1 M NaOH (0.6 mL). The reaction mixture was stirred at room temperature for 1 h, at which point LC-MS analysis revealed that the deprotection of the methyl ester was complete. The reaction mixture was diluted with Dowex® 50WX8 (H + The mixture was neutralized by addition of 1H) resin, filtered and concentrated under reduced pressure. The intermediate crude product 11″ was used directly in the next step without further purification.

[0360] To the intermediate crude compound 11″ was added TFA (1.5 mL). The solution was stirred at room temperature for 1 h until the reaction was complete as evidenced by LC-MS. TFA was removed by rotary evaporation under reduced pressure, treated with diethyl ether (3×2 mL) and dried under vacuum to give compound 12″ as a gummy product. The overall yield over the two steps was 79%.

[0361] Synthesis of compound 13″: To a solution of compound 12″ (4 mg, 5.86 μmol) in DMSO (200 μL), sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate and CBTF (2.60 mg, 6.15 μmol, 1.05 equiv.) were added, followed by triethylamine (8.20 μL, 58.59 μmol, 10.0 equiv.) with stirring at room temperature under argon for 10-15 min. The progress of the reaction was monitored by TLC and LC-MS. After completion of the reaction, compound 13″ was used directly in the next step without any purification (in case of conjugation with Fc protein, it is better to purify using silica gel column).

[0362] For LC-MS evaluation, the conditions were as follows: Column: XBridge BEH C18 Column, 130 Å, 3.5 μm, 3 mm × 100 mm; Mobile phase: A: 20 mM ammonium bicarbonate buffer, pH 7; B: acetonitrile (HPLC grade); Method run: 5–95% B, 7 min, 0.75 mL / min.

[0363] WT-Fc Expression, Purification, and QC Analysis: IgG1 C H 2-C H The 3 wild type was synthesized and subcloned into an expression vector using known methods. Validated plasmid DNA was prepared and used to transiently transfect CHO-S cells. Five to six days after transfection, the cell suspension was centrifuged at 8,000 rpm for 30 min and the supernatant fraction was collected. IgG1 Fc wild type (WT-Fc) was purified by Protein A affinity chromatography.

[0364] Briefly, culture supernatants were passed through a 0.22 μm filter and then loaded onto a polypropylene column packed with Protein A high capacity agarose resin. The resulting flow-through was collected and passed through the column two more times, after which any unbound proteins were washed off with >10 CV (column volume) of 1× PBS. WT-Fc was eluted with 3 ml of 100 mM citrate buffer (pH 3.0) and immediately neutralized with 1 ml of 1 M Tris (pH 8.0). Samples were buffer exchanged into 1× PBS using an Amicon Ultra-4 (Millipore) spin column (10 kDa cutoff), and the purity of the purified samples was assessed on a 4-20% gradient SDS-PAGE gel. The final Fc protein was approximately 26 kDa in monomeric form. The yield was approximately 30-40 mg per 100 ml of culture.

[0365] Final cysteine-based conjugation of Zan-PEG6-Fc-WT: Compound 13″ (10-12 equiv.) was added to purified Fc protein in PBS solution (pH 7.2) with gentle stirring for 10 min at 4° C. After completion of the addition, the reaction mixture was stirred for 12-72 h and the progress of the reaction was monitored by SDS-PAGE and Matrix Assisted Laser Desorption / Ionization (MALDI) mass spectrometry.

[0366] After completion of the reaction, the conjugated crude product was filtered through a molecular weight cut-off (MWCO; 10 kDa, Vivaspin 500, Cat. No. GE28-9322-25) filter and 4 0 The product was purified using centrifugation at 15,000 / RPM, 10 min at 37 °C to remove all unreacted linker and low molecular weight impurities. This process was repeated 3-5 times (at 5 mg / mL concentration) (yield: 60-70%).

[0367] The purity and molecular weight of the zan-PEG6-Fc-WT conjugate were confirmed by SDS-PAGE and MALDI analysis, respectively. The molecular weight of the final product (zan-Fc-WT) was approximately 29 kDa, and that of the Fc protein alone was 26 kDa.

[0368] The above protocol was applied to another cysteine-based conjugate, zan-Fc-DLE, and the product was confirmed by SDS-PAGE and MALDI analysis. As used herein, the term "zan-Fc-WT" is used synonymously with the term "Zan-PEG6-Fc-WT".

[0369] [Example 3] Comparison of single hapten versus dual hapten at 48 and 96 hpi The first group of 6-8 week old female BALB / c mice (n=5 / group) was administered 10 LD 50 Mice were infected with influenza virus A / Puerto Rico / 8 / 1934 (H1N1) (catalog number NR-348, BEI Resources, NIAID, NIH) at 24 hpi. Mice (except for the PBS alone group) were injected intraperitoneally with 8 g / kg human IVIg to achieve humanized titers of anti-DNP and anti-rhamnose antibodies at the time of test article administration. Mice were treated with test articles at 48 hpi. All three test articles were administered as a single intranasal dose of 1.5 umol / kg. Two control groups of mice received PBS as a placebo.

[0370] For evaluation of drug efficacy, mice were monitored by daily weighing for 14 days post-infection and were considered dead when they lost 25% of their body weight or were diagnosed as moribund.

[0371] The results of this experiment are shown in Figures 5 and 6. Figure 5 shows the results of the 100LD 50Figure 6 is a graph of days post-infection vs. survival (%) for mice infected with influenza A H1N1 / PR8 (n=5 / group), depletion of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg (GAMUNEX®-C)) at 24 hpi, and administration of the conjugate at 48 hpi. Figure 6 is a graph of days post-infection vs. body weight (%) for mice treated as described in Figure 5.

[0372] A second group of 6- to 8-week-old female BALB / c mice (n=5 / group) was administered 10 LD 50 Mice were infected with influenza virus A / Puerto Rico / 8 / 1934 (H1N1) (catalog number NR-348, BEI Resources, NIAID, NIH) at 72 hpi (except for the PBS alone group) with 8 g / kg human IVIg intraperitoneally to achieve humanized titers of anti-DNP and anti-rhamnose antibodies at the time of test article administration. Mice were treated with test articles at 96 hpi. All three test articles were administered as a single intranasal dose of 1.5 umol / kg. Two control groups of mice received PBS as a placebo.

[0373] As in previous studies, to assess efficacy, mice were weighed and monitored daily for 14 days post-infection and were considered dead when they lost 25% of their body weight or were diagnosed as moribund.

[0374] The results of this experiment are shown in Figures 7 and 8. Figure 7 shows the results of the 100LD 50 Figure 8 is a graph of days post-infection vs. survival (%) for mice (n=5 / group) infected with influenza A H1N1 / PR8, depletion of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg (GAMUNEX®-C)) at 24 hpi, and administration of the conjugate at 96 hpi. Figure 9 is a graph of days post-infection vs. body weight (%) for mice treated as described in Figure 7.

[0375] [Example 4] Comparison of dual hapten and commercial drug at 48 and 96 hours post-infection The first group of 6-8 week old female BALB / c mice (n=5 / group) was administered 10 LD 50 Mice were infected with influenza virus A / Puerto Rico / 8 / 1934 (H1N1) (catalog no. NR-348, BEI Resources, NIAID, NIH) at 24 hours hpi. Mice (except for the PBS alone group) were injected intraperitoneally with 8 g / kg human IVIg to achieve humanized titers of anti-DNP and anti-rhamnose antibodies at the time of test article administration.

[0376] Treatment of infected mice began 48 hpi. Zan-DNP-rhamnose (compound 24) was administered as a single intranasal dose of 1.5 umol / kg. Tamiflu was administered at a dose of 5 mg / kg twice daily for five consecutive days, and Xofluza was administered at a dose of 1.5 mg / kg twice daily for five days. Two control groups of mice received PBS as a placebo.

[0377] To assess efficacy, mice were monitored by weighing daily for 14 days after infection and were considered dead when they lost 25% of their body weight or were diagnosed as moribund. The results of this experiment are shown in Figures 9 and 10. Figure 9 shows the effect of 100LD 50 Figure 10 is a graph of days post-infection vs. survival (%) for mice infected with influenza A H1N1 / PR8 (n=5 / group), depletion of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg (GAMUNEX®-C)) at 24 hpi, and administration of the conjugate at 48 hpi. Figure 10 is a graph of days post-infection vs. body weight (%) for mice treated as described in Figure 9.

[0378] Separate groups of 6- to 8-week-old female BALB / c mice (n=5 / group) were administered 10LD 50Mice (except for the PBS alone group) were intraperitoneally injected with 8 g / kg human IVIg at 72 hpi to achieve humanized titers of anti-DNP and anti-rhamnose antibodies at the time of test article administration.

[0379] Treatment of infected mice began at 96 hpi. Zan-DNP-rhamnose was administered as a single intranasal dose of 1.5 umol / kg. Tamiflu was administered at a dose of 5 mg / kg twice daily for 5 consecutive days, and Xofluza was administered at a dose of 1.5 mg / kg twice daily for 5 days. Two control groups of mice received PBS as a placebo. For evaluation of drug efficacy, mice were weighed and monitored daily for 14 days after infection and were considered dead when they lost 25% of their body weight or were diagnosed as moribund. The results of this experiment are shown in Figures 11 and 12. Figure 11 shows the results of 100LD 50 1 is a graph of days post-infection vs. survival (%) for mice (n=5 / group) infected with influenza A H1N1 / PR8, depletion of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg (GAMUNEX®-C)) at 24 hpi, and administration of the conjugate at 96 hpi. Figure 12 is a graph of days post-infection vs. body weight (%) for mice treated as described in Figure 11.

[0380] [Example 5] Comparison of dual hapten virus titers with those of the commercial drug at 48 hpi Six to eight-week-old female BALB / c mice (n=5 / group) were administered 10LD 50The mice were infected with influenza virus A / Puerto Rico / 8 / 1934 (H1N1) (catalog number NR-348, BEI Resources, NIAID, NIH) from the University of Illinois. Mice (except for the PBS alone group) were intraperitoneally injected with 8 g / kg human IVIg 24 hours prior to test article (TA) administration to achieve humanized titers of anti-DNP and anti-rhamnose antibodies at the time of test article administration.

[0381] Treatment of infected mice was initiated either 48 hpi or 96 hpi. Zan-DNP-rhamnose (ZDR) (compound 24), zan-DNP (ZD), and zan-rhamnose (ZR) were administered as a single intranasal dose of 1.5 μmol / kg. Tamiflu was administered at a dose of 5 mg / kg twice daily for five consecutive days, and Xofluza was administered at a dose of 1.5 mg / kg twice daily for five days. Two control groups of mice were administered PBS as a placebo.

[0382] To evaluate the rate of viral titer reduction within 24 hours after TA administration, two mice from each cohort were sacrificed by CO2 asphyxiation 24 hours after TA administration, and their lungs were removed and immediately homogenized using a gentleMACS Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Viral titers from lung homogenates were measured by real-time reverse transcription polymerase chain reaction (RT-PCR). RNA was extracted from the homogenates using a Quick-RNA™ Microprep Kit (Zymo Research Corporation, Irvine, California). cDNA synthesis and reverse transcription were performed according to standard protocols. A primer / probe set was synthesized to recognize two highly conserved regions of the influenza matrix (M) gene. To construct a standard curve for the calculation of viral titers, 10-fold dilutions of influenza virus A / Puerto Rico / 8 / 1934 (H1N1) stock solutions with known viral titers were run in parallel with the lung homogenates.

[0383] The results of this experiment are shown in Figures 13A, 13B, 14A, and 14B. Figure 13A shows the viral titers (fold change) for the conjugate (compound 24 = ZDR and zan-DNP = ZD) and the commercial drug (Xofluxa = XO and Tamiflu = TAMI) 48 hpi. Figure 13B shows the viral titers (fold change) for the conjugate (compound 24 = ZDR and zan-DNP = ZD) and the commercial drug (Xofluxa = XO and Tamiflu = TAMI) 96 hpi. Figure 14A shows the viral titers (fold change) for the conjugate (compound 24 = ZDR and zan-DNP = ZD) and the commercial drug (Xofluxa = XO and Tamiflu = TAMI) 48 hpi. FIG. 14B shows the viral titers (fold change) for the conjugates (compound 24=ZDR and zan-DNP=ZD) and commercial drugs (Xofluxa=XO and Tamiflu=TAMI) 96 hpi.

[0384] [Example 6] Oral administration of compound 24 Female Balb / c mice aged 6–8 weeks (5 mice / group) were treated with 100×LD 50 Mice were infected with influenza A / H1N1 / PR8 / 1934. Human IVIg was used as a source of anti-hapten antibodies and was injected at a dose of 8 g / kg 24 hours before drug administration. The test substance, zanamivir-DNP-rhamnose (compound 24), was administered at a single dose of 1.5 or 4.5 μmol / kg via intravenous (IV) and oral routes 48 hours after infection. Mice were weighed and monitored daily for 14 days after infection and considered dead when they lost 25% of their body weight or were diagnosed as moribund.

[0385] When administered orally, survival dropped to 60% at the same dose of test substance and increased to 80% at a 3-fold higher dose. Figure 15A is a graph of days post-infection vs. % survival. Figure 15B is a graph of days post-infection vs. % body weight.

[0386] [Example 7] Quantification of lung viral titers Female Balb / c mice aged 6–8 weeks (2 mice / group) were incubated at 100 × MLD 50 Mice were inoculated intranasally (IN) with influenza A / H1N1 / PR8 / 1934 and then treated 48 hours later with a single IN / IV / OG / SC dose of 1.5 μmol / kg (2.6 mg / kg) or 4.5 μmol / kg (7.8 mg / kg) of Compound 24 or PBS. Mice were euthanized 3 days after infection and their lungs were removed and immediately homogenized using a gentleMACS Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Viral titers from lung homogenates were measured by real-time RT-PCR. Ribonucleic acid (RNA) was extracted from the homogenates using a Quick-RNA™ Microprep Kit (Zymo Research Corporation, Irvine, California). Equal amounts of RNA were used for qrt-PCR using the One Step PrimeScript™ RT-PCR Kit (Takara Bio, Inc., Kusatsu, Japan) according to the manufacturer's protocol. Primer / probe sets were synthesized to recognize two highly conserved regions of the influenza matrix (M) gene. To construct a standard curve for the calculation of viral titers, 10-fold dilutions of influenza virus A / Puerto Rico / 8 / 1934 (H1N1) stock solutions with known viral titers were run in parallel with the lung homogenates.

[0387] Oral administration of compound 24 in mice did not reduce virus titers as rapidly as intranasal or IV administration of the same or three-fold higher doses. Due to reduced bioavailability, oral administration resulted in a slower reduction in virus titers in the lungs compared to the IV and IN routes of administration.

[0388] Figure 16 is a bar graph of route of administration (SC=subcutaneous; OG=oral gavage; IV=intravenous; IN=nasal; PBS=phosphate buffered saline) versus viral titer (PFU / ml per ng of RNA). Figure 17 is a bar graph of OG and PBS versus viral titer (PFU / ml per ng of RNA).

[0389] [Example 8] Pharmacokinetics of Oral Compound 24 Six- to eight-week-old female Balb / c mice (3 mice / group) were administered a single intravenous (IV) or oral dose of 1.5 μmol / kg (2.6 mg.kg), 1.5 μmol / kg (2.6 mg / kg), or 13.5 μmol / kg (23.4 mg / kg) of compound 24, and blood samples were collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h after drug administration. Plasma concentrations of compound 24 were determined by liquid chromatography tandem mass spectrometry (LC-MS / MS). IVIg was administered 24 h prior to drug administration as a source of anti-hapten antibodies. Oral administration resulted in a significant increase in C compared to IV, even at a 9-fold higher dose. max (maximum plasma concentration) was significantly lower, but the half-life (t 1 / 2 ) was extended from approximately 0.4 to 4 hours at a 9-fold higher dose.

[0390] Figure 18 is a graph of time (h=hours) versus concentration (ng / mL) for a single IV dose of 1.5 μmol / kg (n=3 mice / time point). Figure 19 is a graph of time (h=hours) versus concentration (ng / mL) for a single oral dose of 13.5 μmol / kg (n=3 mice / time point).

[0391] [Example 9] Viral load and mouse treatment studies with increasing frequency of oral dosing Female Balb / c mice aged 6–8 weeks (5 mice / group) were treated with 100×LD 50Mice were infected with influenza A / H1N1 / PR8 / 1934. Human IVIg was used as a source of anti-hapten antibodies and was injected at a dose of 8g / kg 24 hours before drug administration. The test substance, compound 24, was administered at 48 hpi in a single dose of 1.5 μmol / kg zan-DNP-rhamnose (IV) or two doses of 4.5 umol / kg zan-DNP-rhamnose (oral). Mice were weighed and monitored daily for 14 days after infection and considered dead when they lost 25% of their body weight or were diagnosed as moribund.

[0392] Increasing the dosing frequency to two doses 12 hours apart resulted in 100% survival (compared to 80% survival with a single dose of 4.5 μmol / kg zan-DNP administered orally). Figure 20A is a graph of days post-infection vs. % survival. Figure 20B is a graph of days post-infection vs. % body weight.

[0393] [Example 10] Quantification of lung viral titers Female Balb / c mice aged 6–8 weeks (2 mice / group) were incubated at 100 × MLD 50Mice were inoculated IN with influenza A / H1N1 / PR8 / 1934 and then treated 48 hours later with a single IV dose of 1.5 μmol / kg (2.6 mg / kg) or two oral doses of 4.5 μmol / kg (7.8 mg / kg) (12 hours apart) of Compound 24 or PBS. Mice were euthanized 3, 5, and 8 days after infection, and their lungs were removed and immediately snap frozen in RNA lysis buffer. Lung homogenates were prepared using a gentleMACS Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Viral titers from the lung homogenates were measured by qrT-PCR. RNA was extracted from the homogenates using a Quick-RNA™ Microprep Kit (Zymo Research Corporation, Irvine, California). Equal amounts of RNA were used for qrt-PCR using the One Step PrimeScript™ RT-PCR Kit (Takara Bio, Inc., Kusatsu, Japan) according to the manufacturer's protocol. Primer / probe sets were synthesized to recognize two highly conserved regions of the influenza matrix (M) gene. To construct a standard curve for the calculation of viral titers, 10-fold dilutions of influenza virus A / Puerto Rico / 8 / 1934 (H1N1) stock solutions with known viral titers were run in parallel with the lung homogenates.

[0394] Oral administration reduced the viral titer at a slower rate than IV administration, but was able to completely eliminate the viral titer by day 8 after infection. Both IV and oral administration of compound 24 could completely eliminate the viral infection from the lungs, with the IV route acting more quickly than the oral route. Figure 21 is a graph of viral titer (PFU / ml per ng of RNA) versus days after infection for PBS and IV and oral (OG=oral gavage) administration of zanamivir-DNP-rhamnose.

[0395] [Example 11] Efficacy comparison of compound 24 with standard of care (SOC) (Treatment 96hpi) Female Balb / c mice aged 6–8 weeks (5 mice / group) were treated with 10×LD 50 Influenza A / H1N1 / PR8 / 1934 (day 0). A control group of healthy mice (not infected on day 0) was also tested as a control.

[0396] Human IVIg was used as a source of anti-hapten antibodies and was injected at an optimized dose of 6 g / kg 24 hours prior to drug administration to each cohort. Test substances and SOC were administered at 96 hpi with a single IV dose of 2.6 mg / kg of compound 24 (C.24(A) in Figures 22 and 23), a single oral dose of 23.4 mg / kg of compound 24 (C.25(B) in Figures 22 and 23), a single oral dose of 12.3 mg / kg baloxavir marboxil (Xofluza®) (Xofluza (C) in Figures 22 and 23), 5 mg / kg oseltamivir phosphate (Tamiflu®) orally twice daily for 5 days (Tamiflu (D) in Figures 22 and 23), or PBS (E in Figures 22 and 23) (healthy mouse data not shown in Figures 22 and 23). Mice were monitored by daily weighing for 14 days post-infection and were considered dead when they lost 25% of their body weight or were deemed moribund.

[0397] Both single dose IV (A) and oral administration (B) of compound 24 resulted in 100% survival of virus-infected mice, which was not achieved by either of the two SCO drugs tested. Compound 24 outperformed the current standard of care antiviral when treated at 96 hpi. Figure 22 is a graph of days post-infection vs. survival (%). Figure 23 is a graph of days post-infection vs. body weight (%). (Compound 24=C24 in Figures 23 and 24).

[0398] Twenty-four hours after drug administration (120 hpi), mice were anesthetized with isoflurane followed by cervical dislocation. Lung tissues were fixed in 10% neutral buffered formalin (NBF), embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and then imaged at 10x magnification on a Nikon Eclipse light microscope. Qualitative histological analysis of the lungs was performed and images are shown in Figure 33. Untreated virus-infected mice showed signs of diffuse alveolar damage, pulmonary edema, and hyperinfiltration of inflammatory cells, whereas mice treated with compound 24 either intravenously or orally showed a significant reduction in virus-induced histopathological changes in lung tissue when compared to the untreated group.

[0399] In parallel, two cohorts of healthy mice were injected with compound 24 IV and compound 24 OG, respectively, and another cohort of mice was not intervened. 24 hours after drug administration, mice were anesthetized with isoflurane, followed by cervical dislocation. Lung, kidney, liver, stomach, and small intestine tissues were fixed in 10% neutral buffered formalin (NBF), embedded in paraffin, sectioned, stained with H&E (except kidney, kidney tissue was stained with periodic acid Schiff (PAS)), and then imaged at 10x magnification on a Nikon Eclipse light microscope. Qualitative histological analysis of lungs was performed, and the images are shown in Figure 33. No abnormal changes in tissue morphology were observed in mice injected with compound 24 when compared with the non-intervention group.

[0400] [Example 12] Comparison of efficacy of compound 24 with monohaptens (Treatment 96hpi) A targeted therapeutic strategy with a dual mechanism of action that induces a host immune response against the virus and virus-infected cells is disclosed. As a targeting ligand, the neuraminidase inhibitor zanamivir (as a fragment) is deployed. Neuraminidase is present both on the influenza virus envelope and on the surface of infected cells. The conjugate containing the zanamivir fragment is linked to a hapten that binds to antibodies naturally occurring in humans. Once recruited, these anti-hapten antibodies bind and activate the innate immune system against the virus and virus-infected cells.

[0401] Female Balb / c mice aged 6–8 weeks (5 mice / group) were treated with 10×LD 50 Mice were infected with influenza A / H1N1 / PR8 / 1934 (day 0). Human IVIg was used as a source of anti-hapten antibodies and was injected at an optimized dose of 6 g / kg 24 hours prior to drug administration. Test substances were administered at 96 hpi with a single IV dose of 1.5 μmol / kg of (a) compound 24 ((A) in Figures 29A and 29B); (b) zanamivir-DNP (monohapten conjugate; (B) in Figures 29A and 29B); (c) zanamivir-rhamnose (monohapten conjugate; (C) in Figures 29A and 29B); (d) zanamivir ((D) in Figures 29A and 29B); or (e) PBS (control) ((E) in Figures 29A and 29B). Mice were monitored by daily weighing for 14 days post-infection and were considered dead when they lost 25% of their body weight or were deemed moribund.

[0402] When tested in BALB / c mice supplemented with IVIg and infected with influenza A virus (H1N1, A / Puerto Rico / 8 / 1934), the zanamivir-dual hapten conjugate (compound 24) showed superior antiviral activity at both early and late stages of infection compared to the monohapten conjugate. Furthermore, the dual hapten conjugate showed better activity at late stages of infection compared to the monohapten conjugate at a single dose. Thus, compound 24 can treat both early and late stages of influenza infection.

[0403] [Example 13] Comparison of lung titers at 24 hours Female Balb / c mice aged 6–8 weeks (5 mice / group) were treated with 10×LD 50 of influenza A / H1N1 / PR8 / 1934 and treated 48 hours later with (a) a single IV dose of 1.5 μmol / kg of Compound 24; (b) a single OG dose of 13.5 μmol / kg of Compound 24; (c) a single OG dose of 5 mg / kg Tamiflu®; (d) a single OG dose of 10 mg / kg Xofluza®; or (e) a single IV dose of 100 μL PBS.

[0404] 24 hours after treatment, virus titers from lung homogenates were measured by real-time RT-PCR, the results shown in FIG. 24 showed the lowest titers with orally administered Compound 24 (Compound 24=C.24).

[0405] [Example 14] Comparison of administration routes for Compound 24 As shown in Figure 25, Balb / c mice were exposed to infection on day 0. Four days after infection challenge, compound 24 was administered by three different routes, intranasal, IV, and oral, as indicated, with oral being the highest dose at 13.5 μmol / kg, and the other doses at 1.5 μmol / kg. The results show that survival rates were 100% after 14 days for all three compound 24 cohorts, while mice treated with PBS did not survive beyond day 10. A graph of infection / body weight % is shown in Figure 26.

[0406] [Example 15] Antibody-mediated effector functions in vitro To test the proof-of-concept of the mechanism of action of compound 24 in vitro, antibody-dependent cellular cytotoxicity (ADCC) assay and complement-dependent cytotoxicity (CDC) assay were performed. ADCC assay was performed with influenza virus neuraminidase (N1) transfected (NA-HEK) and wild-type (WT) human embryonic kidney (HEK293) cells using an ADCC kit (Promega, Catalog No. G7010; Promega Corporation, Madison, WI). For this purpose, cells were plated in triplicate (100 μl, 5000 cells / well) in 96-well black-walled plates (Corning Life Sciences, Corning, NY) and then treated with serial dilutions of compound 24 in the presence or absence of a 100-fold excess of zanamivir. After incubation at 37° C. for 2 hours, human IVIg was added to each well and the plate was incubated for an additional 30 minutes at 37° C. Finally, ADCC effector cells were added at 75,000 cells / well and incubated overnight at 37° C. under 5% CO 2 .

[0407] The amount of firefly luciferase produced by the ADCC effector cells was then quantified using Bio-Glo™ Luciferase Assay Reagent (included in the kit). Luminescence was measured using a Synergy Neo2 HTS Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT).

[0408] Analysis of virus-infected cell killing by the complement system was performed by complement-dependent cytotoxicity (CDC) assay. N1-transfected (NA-HEK) HEK293 cells and wild-type (WT) HEK293 cells were harvested and plated in triplicate (100 μl, 5000 cells / well) in 96-well black-walled plates (Corning Life Sciences, Corning, NY), then treated with serial dilutions of compound 24 in the presence or absence of 100-fold excess zanamivir. (Zanamivir is a free drug that competes with compound 24 when used in excess). After incubation at 37°C for 2 hours, human IVIg and human serum were added to each well, and the plate was incubated overnight at 37°C under 5% CO2. CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (Promega Corporation, Madison, WI) was used to measure cell viability.

[0409] Compound 24, in the presence of antibodies, induced antibody-mediated effector function in vivo with high potency and selectivity by interacting with Fc receptors and complement proteins expressed on effector cells. Figures 27A and 27B show graphs of compound 24 concentration (nM) versus ADCC% and CDC%, respectively.

[0410] [Example 16] Dose Escalation of IV Compound 24 Female Balb / c mice aged 6–8 weeks (5 mice / group) were treated with 10×LD 50Mice were infected with influenza A / H1N1 / PR8 / 1934 (day 0). Human IVIg was used as a source of anti-hapten antibodies and was injected at an optimized dose of 6 g / kg 24 hours before drug administration. Mice in each test group were administered a single IV dose of (a) 0.17 μmol / kg compound 24 ((A) in Figure 28A and Figure 28B); (b) 0.5 μmol / kg compound 24 ((B) in Figure 28A and Figure 28B); (c) 1.5 μmol / kg compound 24 ((C) in Figure 28A and Figure 28B); (d) 4.5 μmol / kg compound 24 ((D) in Figure 28A and Figure 28B); and (e) PBS ((E) in Figure 28A and Figure 28B) at 96 hpi. Mice were monitored by daily weighing for 14 days post-infection and were considered dead when they lost 25% of their body weight or were deemed moribund.

[0411] A single dose of 1.5 μmol / kg IV of Compound 24 (C) and a single dose of 4.5 μmol / kg IV of Compound 24 (D) both resulted in 100% survival of mice that was not achieved with other doses tested or with the control (PBS). Figures 28A and 28B show graphs of days post-infection vs. survival (%) and days post-infection vs. body weight (%), respectively.

[0412] [Example 17] Effective against multiple flu strains On day 0, 6- to 8-week-old female Balb / c mice (5 mice / group) were inoculated with 10 × LD 50Mice were infected with influenza A / California / 07 / 2009(H1N1)pdm09, influenza A / Wisconsin / 67 / 2005, or influenza B / Florida / 04 / 2006. Mice infected with influenza A / California / 07 / 2009(H1N1)pdm09 were administered a single IV dose of 1.5 μmol / kg of compound 24 ((A) in Figures 30A and 30B) or PBS ((D) in Figures 30A and 30B) at 96 hpi. Mice infected with influenza A / Wisconsin / 67 / 2005 were administered a single IV dose of 1.5 μmol / kg of compound 24 ((B) in Figures 30A and 30B) or PBS ((E) in Figures 30A and 30B) at 96 hpi. Mice infected with influenza B / Florida / 04 / 2006 were administered a single IV dose of 1.5 μmol / kg of compound 24 ((C) in Figures 30A and 30B) or PBS ((F) in Figures 30A and 30B) at 96 hpi.

[0413] Compound 24 was equally effective against all strains of flu tested, with survival rates of 100% after 14 days for all three compound 24 cohorts, whereas no mice treated with PBS survived beyond day 11.

[0414] [Example 18] Inflammatory cytokine and chemokine levels Female Balb / c mice aged 6–8 weeks (5 mice / group) were treated with 10×LD 50The mice were infected with influenza A / H1N1 / PR8 / 1934 (day 0). Mice in each test group were administered a single dose of one of the following treatments at 48 hpi or 96 hpi: 100 μL PBS IV (A); 2.6 mg / kg Compound 24 by single dose IV (B); 23.4 mg / kg Compound 24 by single dose OG (C); 12.3 mg / kg Xofluza® by single dose OG (D); and 5 mg / kg Tamiflu® by OG bid for 5 days (E). Two groups of mice were not infected with virus but were administered Compound 24 by IV at 96 hpi (F) (compared to the other groups), and the third group of mice was not infected or given any treatment (G). Cytokines and chemokines from each group were measured in lung tissue samples by BioLegend's LEGENDplex™ bead-based immunoassays to determine cytokine and chemokine levels using standard protocols provided by the manufacturer (BioLegend, San Diego, CA).

[0415] Figure 31 shows a graph of the specific cytokine and chemokine expression levels in the lungs measured for each group in both the 48hpi and 96hpi treatment groups. When no cytokine or chemokine level is shown for the control group in the graph, the level was undetectable. Compound 24, whether administered orally or intravenously, did not induce a cytokine storm in the lungs in either case, but rather prevented the occurrence of a cytokine storm by rapidly eliminating the root cause of the cytokine storm (i.e., viral infection). These data support that administration of compound 24 can prevent lung inflammation and damage caused by influenza virus infection.

[0416] Serum cytokine and chemokine levels are also measured to evaluate whether or not subject has systemic inflammation. Figure 32 shows the graph of the serum specific cytokine and chemokine expression levels measured for each group in both 48hpi treatment group and 96hpi treatment group, where A is PBS control, B is compound 24 treatment group with single dose IV, and C is compound 24 treatment group with single dose OG.

[0417] All Compound 24 treatment groups exhibited lower levels of inflammatory cytokines compared to other treatment groups and controls.

[0418] [Example 19] Synthesis of folic acid dual hapten conjugate (compound 150) FIG. 35 shows a synthetic scheme for the synthesis of compound 150.

[0419] Synthesis of Compound 3': Compounds 1' and 2' were prepared according to the procedure described in Ref Chemistry - An Asian Journal 7(2): 272-276 (Theresa Kueckmann et al. eds.) (2012). Then, to a solution of 1-chloro-2,4-dinitrobenzene (compound 1') (0.5 g, 2.47 mmol) and 3-(2-aminoethoxy)propanoic acid (compound 2', 0.33 g, 2.47 mmol) dissolved in EtOH (25 mL), TEA (1.38 mL, 9.87 mmol) was added. The reaction mixture was heated to 55° C. for 16 h and the progress of the reaction was monitored by LC-MS. After completion of the reaction was confirmed by disappearance of one of the starting materials (i.e., dinitrobenzene), the reaction mixture was cooled and concentrated under reduced pressure. The crude mixture was purified by flash chromatography on a Teledyne CombiFlash Rf+Lumen (silica gel, 12 g column, 0-20% methanol in DCM) to give compound 3' as a yellow solid (90% yield). LC-MS [M+H]+=300.24 (see Figure 36).

[0420] Synthesis of compound 5': To a solution of 3-(2-((2,4-dinitrophenyl)amino)ethoxy)propanoic acid (compound 3', 0.1 g, 0.33 mmol) in dimethyl sulfoxide (2 mL) under argon atmosphere, HATU (0.11 g, 0.28 mmol, 0.85 equiv.) was added followed by DIPEA (0.29 mL, 1.67 mmol, 5.0 equiv.) and stirred at room temperature for 10 min. To this reaction mixture was added Fmoc-Lys-OH.HCl (obtained from Chem-Impex International, Wood Dale, IL; compound 4', 0.11 g, 0.27 mmol, 0.8 equiv.) and stirred at room temperature for 2-3 h, and the progress of the reaction was monitored by LC-MS.

[0421] After the reaction was confirmed to be complete by LC-MS, the reaction mixture was quenched by adding water and extracting with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The resulting crude material was purified by silica gel (4 g) column chromatography on a Teledyne CombiFlash Rf+ Lumen (0-10% methanol in DCM) and fractions were analyzed by LC-MS. The solvent was evaporated from the combined pure fractions using a rotary evaporator under low vacuum, and compound 5' was isolated in 70% yield. LC-MS [M+H]+=650.67 (see FIG. 37).

[0422] Synthesis of 2,3,4-tetra-O-acetyl-α-L-rhamnose (compound 7'): Compound 7' was prepared according to the procedure described in De Coen et al., Synthetic rhamnose glycopolymer cell-surface receptor for endogenous antibody recruitment, Biomacromolecules 21(2): 793-802 (2020). Briefly, α-L-rhamnose monohydrate (6, 1.0 g, 5.49 mmol) was dissolved in 9.2 mL of anhydrous pyridine. The solution was stirred in an ice bath, purged with nitrogen, and then acetic anhydride (4.15 mL, 43.92 mmol, 8.0 equiv.) was added dropwise over 15 min by maintaining the internal temperature below 10 °C. The reaction was allowed to warm slowly to room temperature over 2 hours, and the progress of the reaction was monitored by TLC (hexane / EtOAc, 65:35) and LC-MS, which showed complete consumption of α-L-rhamnose monohydrate after 20 hours of reaction under inert atmosphere. The reaction mixture was poured into ethyl acetate and extracted twice with 1.0 M HCl. The combined organic layers were washed with saturated sodium carbonate solution, water, and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting crude oily product, compound 7' (98% yield), was used in the next step. LC-MS [M+H]+ = 333.32, and / or LC-MS [M+H2O] = 350.32.

[0423] Synthesis of Compound 9': Compound 9' was prepared according to the procedure described in De Coen et al. (2020) supra. Briefly, 1,2,3,4-tetra-O-acetyl-α-L-rhamnose (7, 0.50 g, 1.50 mmol) was dissolved in DCM (7.5 mL) and then -H2N-PEG4-OH (obtained from BroadPharm, San Diego, CA; 8, 0.35 g, 1.81 mmol, 1.2 equiv.) was added under an inert atmosphere. The reaction flask was placed in an ice bath and boron trifluoride diethyl etherate (0.56 mL, 4.51 mmol, 3.0 equiv.) was added dropwise over 30 min at 4°C. The reaction mixture was stirred at ice bath temperature for 2 h and then the reaction was allowed to warm to room temperature. The progress of the reaction was monitored by TLC (hexane / EtOAc, 30:70, Rf=0.30) and LC-MS, which showed complete consumption of 1,2,3,4-tetra-O-acetyl-α-L-rhamnose (compound 7') after 16 h of reaction. The reaction mixture was poured into ice water and extracted with DCM (3x10 mL). The combined organic layers were washed twice with saturated sodium bicarbonate solution, water, and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting crude material was purified by silica gel (12 g) column chromatography on a Teledyne CombiFlash Rf+ Lumen (0-10% methanol in DCM) to isolate the product (compound 9') (90% yield). LC-MS [M+H]+=466.51.

[0424] Synthesis of compound 10': To a solution of acid (compound 5', 0.06 g, 0.09 mmol) and rhamnose(OAc)3-PEG4-NH2 (compound 9', 0.04 g, 0.09 mmol, 1.0 equiv.) in dimethylsulfoxide (1.5 mL) was added PyBOP (0.05 g, 0.10 mmol, 1.1 equiv.) followed by DIPEA (0.081 mL, 0.46 mmol, 5.0 equiv.) at room temperature under argon atmosphere. The progress of the reaction was monitored by LC / MS. After the reaction was complete as confirmed by LC-MS, the reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The obtained crude material was purified by silica gel (4 g) column chromatography using Teledyne CombiFlash Rf+ Lumen (0-10% MeOH in dichloromethane) to isolate the product (compound 10') (93% yield). LC-MS [M+H]+=1098.15 (see FIG. 38).

[0425] Deprotection of -Fmoc group to synthesize compound 11': To compound 10' (0.01 g, 0.01 mmol) in dry DCM (0.2 ml) was added DEA (100 uL) under argon at room temperature. The solution was stirred at room temperature for 1 hour until the reaction was complete as demonstrated by LC-MS. DEA was removed by rotary evaporation under reduced pressure, and the crude product was precipitated in diethyl ether to obtain a yellow solid in quantitative yield and used in the next step without further purification. LC-MS [M+H]+=875.91 (see FIG. 39).

[0426] Synthesis of compound 13': To a solution of DBCO-NHS (obtained from BroadPharm, San Diego, CA; compound 12', 0.012 g, 0.029 mmol, 1.05 equiv.) and compound 11' (0.024 g, 0.027 mmol, 1.0 equiv.) dissolved in DCM:DMSO (10:1, 0.6 mL) under argon was added DIPEA (0.048 μl, 0.27 mmol, 10 equiv.) dropwise. The reaction mixture was stirred at room temperature for 2-3 h and the progress of the reaction was monitored by LC-MS. After completion of the reaction (determined by complete consumption of compound 11'), the solvent was evaporated by rotary evaporator under reduced pressure, and the crude material was purified by silica gel (4 g) column chromatography by Teledyne CombiFlash Rf+ Lumen (0-10% methanol in DCM) to obtain compound 13' as a yellowish solid (93% yield). LC-MS [M+H]+=1163.2. (See Figure 40).

[0427] Synthesis of intermediate 14': Compound 13' (0.025 g, 0.022 mmol) was dissolved in anhydrous MeOH (1 mL) and treated dropwise with 0.5 M NaOH (25 μL). The reaction mixture was stirred at room temperature for 1 h, at which point LC-MS analysis revealed the absence of starting material. The reaction was cooled to room temperature using Dowex® 50WX8 (H + Neutralized by adding 14-methyl-1,1-dichloro-2 ...

[0428] Synthesis of folic acid-NHS ester (compound 16): To a solution of compound 15' (0.50 g, 1.133 mmol) and N-hydroxysuccinimide (0.143 g, 1.246 mmol, 1.10 equiv.) in DMSO (12 mL) under argon at room temperature, DCC (0.281 g, 1.359 mmol, 1.2 equiv.) was added and the solution was stirred for 12 h. The progress of the reaction was monitored by LC / MS. After the completion of the reaction was confirmed by LC-MS, the reaction mixture was precipitated in acetone (15 times) and the product was isolated by centrifugation. The crude product was further washed with acetone (2 times 50 mL) and EtOAc (1 time 30 mL), dried under vacuum, and the crude product (compound 16) was used in the next step without further purification.

[0429] Synthesis of folate-PEG6-azide (compound 18'): To a solution of folate-NHS ester (compound 16, 0.05 g, 0.093 mmol) in dry DMSO (1.0 mL), N3-PEG6-NH2 (compound 17'; 0.032 g, 0.093 mmol, 1.05 eq., obtained from BroadPharm, San Diego, CA) was added at room temperature under argon, followed by DIPEA (0.081 mL, 0.46 mmol, 5.0 eq.) and stirred for 12 hours. The progress of the reaction was monitored by LC / MS. After the completion of the reaction was confirmed by LC / MS, the reaction mixture was precipitated by adding diethyl ether and centrifuged. The yellow precipitate was collected and washed with diethyl ether (2×15 mL) and the crude mass was purified by preparative HPLC on a C18 column (5-95% B over 60 min, flow rate 7 mL / min; B: acetonitrile; A: 20 mM NH4OAc, pH 7 buffer, UV@280 nm) to give compound 18'. LC-MS [M+H]+=774.83 (see Figure 42).

[0430] Synthesis of Compound 150: To a solution of Compound 18' (10 mg, 0.013 mmol) and Compound 14' (13.4 mg, 0.013 mmol, 1.0 equiv.) in dry DMSO (1.0 mL) under argon, DIPEA (12 μl, 0.065 mmol, 5.0 equiv.) was added dropwise. The reaction mixture was stirred at room temperature for 2-4 h and the progress of the reaction was monitored by LC-MS. After completion of the reaction, the crude product was purified by preparative HPLC on a C18 column (5-95% B over 60 min, flow rate 7 mL / min; B: acetonitrile; A: 20 mM NHOAc, pH 7 buffer, UV@360 nm and 280 nm) to obtain the final compound Compound 150 (yield 24%, purity: 97% by HPLC, and LC-MS [M+H]+=1810.93) (see FIG. 43).

[0431] [Example 20] In vitro functional assays with compound 150 Folate receptor-expressing mouse lung cancer cells (cell line M109) were seeded in 96-well plates at a density of 500 cells / well and allowed to adhere overnight at 37° C. Cells were then incubated with serial dilutions of folate dual hapten conjugate compound 150 for 2 hours at 37° C., after which human serum was added to assess the effect, if any, on complement-dependent cytotoxicity (CDC).

[0432] The results are shown in Figure 44 and confirm that addition of compound 150 induced CDC, likely by recruiting anti-hapten antibodies on the cancer cell surface and subsequent activation by antibodies of the complement system in human serum. A 100-fold excess of folate glucosamine was used as a competitor in the control set to confirm that the observed immunogenic effect was indeed due to binding of the folate receptor on the target cells with the folate moiety on the dual hapten conjugate (identified as M109_comp in Figure 44).

[0433] In addition, cancer cells expressing folate receptors (4T1 (mouse breast cancer cells), MDA-MB-231 (human breast cancer cells), M109 (mouse lung cancer cells)), and THO-1 (AML model) were assayed for ADCC activity of compound 150. After 2 hours of incubation with serial dilutions of compound 150, the cells were then incubated with IVIg for 2 hours at 37°C and incubated with human Fc-γ-RIII-expressing effector cells overnight at 37°C, 5% CO2. The results are shown in Figures 45A-45D and confirm that compound 150 induced ADCC by recruiting anti-hapten antibodies on the cancer cell surface and subsequently activating Fc-γ-RIII receptors expressed on effector cells.

[0434] [Example 21] In vivo efficacy of compound 150 in a mouse lung cancer model Female Balb / c mice aged 6–8 weeks were given 2 × 10 6 M109 mouse lung cancer cell line (mouse lung cancer cells expressing folate receptors) was inoculated at 10 cells / mouse. Ten days after inoculation, the tumor size was approximately 50 mm. 3 If the tumor reached 100 μL / mouse, treatment was initiated with the following test substances: (a) 100 μL / mouse PBS alone; (b) SOC treatment (i.e., 200 μg / mouse 3 days / week anti-PD1 + chemotherapy (50 mg / kg carboplatin intraperitoneal injection on day 18 after tumor inoculation + 36 mg / kg paclitaxel intravenous injection on days 18-22 after tumor inoculation)); or (c) 10 nmol / mouse Compound 150 (daily doses over 5 days / week, respectively). Anti-DNP and anti-rhamnose antibodies were also injected intraperitoneally in the PBS and Compound 150 groups on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study period.

[0435] Tumors were measured every other day and mice were sacrificed 25 days after tumor implantation. Figures 46 and 47 show the tumor volume (mm 3) versus days after tumor implantation, and subject body weight percentage versus days after tumor implantation. This data demonstrates that compound 150 inhibits tumor growth in the M109 model, demonstrating its promise as a monotherapy in the treatment of lung cancer.

[0436] [Example 22] Efficacy of Compound 150 in an in vivo study in a human lung cancer (cold tumor) model Six- to eight-week-old female C57BL / 6 mice were given 5 × 10 6 Cells / mouse of the LLC-1 human lung cancer cell line (LLC-1) were inoculated. Ten days after inoculation, the tumor size was approximately 50 mm 3 When the 100 μL / mouse PBS alone was reached, treatment was started with the following test substances: (a) 100 μL / mouse PBS alone; (b) SOC treatment (i.e., 200 μg / mouse anti-PDL1 intraperitoneal injection 3 days / week + 2 Gy / mouse radiation treatment 3 days / week); or (c) 10 nmol / mouse Compound 150 (daily doses for 5 days / week, respectively). On days 10, 14, 17, and 21 after transplantation, the PBS and Compound 150 groups were also intraperitoneally injected with anti-DNP and anti-rhamnose antibodies to achieve humanized titers of anti-hapten antibodies over a period of 2 weeks.

[0437] Tumors were measured every other day and mice were sacrificed 25 days after tumor implantation. Figures 48 and 49 show the tumor volume (mm 3 ) versus days after tumor implantation, and subject body weight percentage versus days after tumor implantation. This data supports that compound 150 may benefit from combination with treatments that transform cold tumors into hot tumors.

[0438] [Example 23] In vivo efficacy of compound 150 in a mouse colorectal cancer model Female Balb / c mice aged 6–8 weeks were given 2 × 10 6 CT26 murine colorectal cancer cell line was implanted at 10 cells / mouse. Ten days after inoculation, tumor size was approximately 50 mm. 3When the tumor reached 100 μL / mouse, treatment was started with the following test substances: (a) 100 μL / mouse PBS alone; (b) SOC treatment (i.e., weekly intraperitoneal injection of Leacovorin 100 mg / kg + weekly 5-FU 50 mg / kg + oxaliplatin 6 mg / kg on days 8 and 10 after tumor inoculation); or (c) 10 nmol / mouse Compound 150 (daily doses for 5 days / week, respectively). On days 10, 14, 17, and 21, the PBS and Compound 150 groups were also intraperitoneally injected with anti-DNP and anti-rhamnose antibodies to achieve humanized titers of anti-hapten antibodies throughout the study period.

[0439] Tumors were measured every other day and mice were sacrificed 25 days after tumor implantation. Figures 50 and 51 show the tumor volume (mm 3 ) versus days after tumor implantation, and subject body weight percentage versus days after tumor implantation. Mice exhibiting greater than 25% weight loss were euthanized as a humane endpoint.

[0440] All mice in the leucovorin + 5-FU + oxaliplatin group died by day 17, whereas all mice in the compound 150 cohort survived and had slower tumor growth rates compared to untreated controls.

[0441] [Example 24] Evaluating multiple combination therapies in a murine lung cancer model Female Balb / c mice aged 6–8 weeks (n=5 per cohort) were given 2–5 × 10 6M109 mouse lung cancer cell line (mouse lung cancer cells expressing folate receptors) of 10 cells was implanted (day 0). On day 11 after implantation, treatment was started with the following test substances: (a) 10 μL / mouse PBS alone; (b) chemotherapy (carboplatin + paclitaxel); (c) 10 nmol / mouse Compound 150 + anti-PD1; (d) 10 nmol / mouse Compound 150 (+Abs); and (e) 10 nmol / mouse Compound 150 + chemotherapy. On days 10, 14, 17, and 21, the PBS and Compound 150 groups were also intraperitoneally injected with anti-DNP and anti-rhamnose antibodies to achieve humanized titers of anti-hapten antibodies throughout the study period. Tumors were measured every other day.

[0442] FIG. 52 shows the tumor volume (mm 3 ) versus days after tumor implantation. Two of five animals in the chemotherapy treatment cohort were tumor-free by day 17 after implantation (all animals in this cohort survived until the end of the study). Three of five animals in the combination therapy of compound 150 + anti-PD1 cohort were tumor-free (one of the remaining animals was not alive at day 22 after implantation). Four of five animals in the combination therapy (compound + chemotherapy) cohort were tumor-free (three of five animals were not alive at day 22 after implantation). This data supports that the combination of compound 150 with anti-PD-1 or chemotherapy significantly enhanced treatment efficacy.

[0443] Another study was performed using the same protocol as described above with the following test substances: (i) 100 μL / mouse PBS alone; (ii) SOC treatment (anti-PD1 + chemotherapy (carboplatin + paclitaxel)); (iii) 10 nmol / mouse Compound 150 + sunitinib; (iv) 10 nmol / mouse Compound 150 (+Abs); and (v) 10 nmol / mouse Compound 150 + folate-toll-like receptor 7 agonist conjugate (FA-TLR7). Anti-DNP and anti-rhamnose antibodies were also injected intraperitoneally in the PBS and Compound 150 groups on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study period.

[0444] FIG. 53 shows the tumor volume (mm 3 ) versus days after tumor implantation. Four of five animals in the SOC treatment cohort were tumor-free (all animals in this cohort survived until the end of the study). One of five animals in the compound 150 + sunitinib combination therapy cohort was tumor-free, but died 22 days after implantation. One of five animals in the compound + FA-TLR7 combination therapy cohort was tumor-free (all animals in this cohort survived until the end of the study). This data confirms that the combination of compound 150 and sunitinib slightly enhanced the antitumor efficacy of treatment, but the combination of compound 150 and FA-TLR7 did not achieve any statistically relevant efficacy improvement at these doses.

[0445] [Example 25] Comparative efficacy of compound 150 in in vivo studies in mouse colorectal cancer Female Balb / c mice aged 6–8 weeks (n=5 per cohort) were given 2 × 10 6 CT-26 mouse colorectal cancer cell line was implanted (day 0) at 10 cells / mouse. Treatment was started 11 days after implantation with the following test substances: (a) PBS alone; (b) Leacovorin+5FU+Oxaliplatin (OXH) (SOC cohort); or (c) 10 nmol / mouse Compound 150 (daily dose, respectively). Treatment was terminated 22 days after implantation. Anti-DNP and anti-rhamnose antibodies were also injected intraperitoneally in the PBS and Compound 150 groups on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study period.

[0446] Tumors were measured every other day and mice were sacrificed 25 days after tumor implantation (all animals survived to the end of the study). Figures 54 and 55 show the tumor volume (mm 3) versus days after tumor implantation, and subject body weight percentage versus days after tumor implantation. Three of five animals in the SOC cohort were tumor-free, and all animals had lost less than 75% of their body weight by day 19 post-implant and were euthanized.

[0447] [Example 26] Evaluating multiple combination therapies in a murine colorectal cancer model Female Balb / c mice aged 6–8 weeks (n=5 per cohort) were given 2–5 × 10 6 CT-26 mouse colorectal cancer cell line of 100 cells was implanted (day 0). On day 11 after implantation, treatment was started with the following test substances: (a) 100 μL / mouse PBS alone; (b) chemotherapy (carboplatin + paclitaxel); (c) 10 nmol / mouse Compound 150 + anti-PD1; (d) 10 nmol / mouse Compound 150 (+Abs); (e) 10 nmol / mouse Compound 150 + chemotherapy; and (f) SOC (Leacovorin + 5FU + oxyplatin). On days 10, 14, 17, and 21, the PBS and Compound 150 groups were also intraperitoneally injected with anti-DNP and anti-rhamnose antibodies to achieve humanized titers of anti-hapten antibodies throughout the study period. Tumors were measured every other day.

[0448] FIG. 56 shows the tumor volume (mm 3 ) versus days after tumor implantation. Four of five animals in the compound 150 + anti-PD1 combination therapy cohort were tumor-free. Three of five animals in the compound 150 + chemotherapy combination therapy cohort were tumor-free. Two of five animals in the chemotherapy (alone) cohort were tumor-free. This data supports that the combination treatment of compound 150 with chemotherapy works better than chemotherapy treatment alone, and that the combination treatment of compound 150 with anti-PD1 works better than SOC.

[0449] Another study was carried out using the same protocol as described above with the following test substances: (i) 100 μL / mouse PBS alone; (ii) Leacovorin+5FU+OXH(SOC); (iii) 10 nmol / mouse Compound 150+Sunitinib; (iv) 10 nmol / mouse Compound 150(+Abs); and (v) 10 nmol / mouse Compound 150+Folic Acid-TLR7 Conjugate. Anti-DNP and anti-rhamnose antibodies were also injected intraperitoneally into the PBS and Compound 150 groups on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study period.

[0450] Figure 57 shows the tumor volume (mm 3 ) versus days after tumor implantation. One of five animals in the combination treatment cohort of compound 150 + sunitinib cohort disappeared from tumor. One of five animals in the combination treatment cohort of compound 150 + FA-TLR7 disappeared from tumor. This data supports that the combination treatment of compound 150 with sunitinib or FA-TLR7 significantly enhances the antitumor efficacy of treatment.

[0451] FIG. 58 shows a graph comparing the data from FIG. 56 and FIG. 57 to highlight the differences between compound 150 and anti-PD1 / sunitinib / chemotherapy combination therapy compared to SOC treatment alone.

[0452] [Example 27] Comparative efficacy of compound 150 in vivo studies in mouse lung cancer model Y856 (cold tumor model) Six to eight week old female C57BL / 6 mice (n=5 per cohort) were given 2 × 10 6Cells of the Y856 mouse lung cancer cell line were implanted (day 0). 11 days after implantation, treatment was started with the following test substances: (a) PBS (100 μL / mouse IV) + anti-DNP / Rham.Ab.; (b) anti-PD1 (200 μg / mouse IP 3 days / week) + carboplatin (50 mg / kg IP on day 18 after tumor inoculation) + paclitaxel (36 mg / kg IV once daily from days 18 to 22 after tumor inoculation); or (c) FDR (compound 150) (10 nmol / mouse once daily) + anti-DNP / Rham.Ab.

[0453] Tumors were measured every other day and mice were sacrificed 25 days after tumor implantation. 3 ) versus days after tumor implantation. Anti-DNP and anti-rhamnose antibodies were also injected intraperitoneally in the PBS and Compound 150 groups on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study period.

[0454] [Example 28] In vivo efficacy of compound 150 in a mouse kidney cancer model Female Balb / c mice aged 6–8 weeks were given 2 × 10 6 Renca cancer cell line was implanted at 100μL / mouse IV. 12 days after implantation, treatment was started with the following test substances: (a) PBS (100μL / mouse IV) + anti-DNP / Rham Ab.; (b) anti-PD1 (200μg / mouse IP 3 days / week) / PD-L1 (200μg / mouse IP 3 days / week) + sunitinib (20mg / kg OG once daily); or (c) FDR (compound 150) (10nmol / mouse once daily) + anti-DNP / Rham Ab. (daily doses, respectively).

[0455] Tumors were measured every other day and mice were sacrificed 25 days after tumor implantation. 3 ) versus days after tumor implantation.

[0456] [Example 29] Immune response assessment Tumors from the above study in Balb / c mice using Y856, LLC-1, and M109 cell lines were minced and digested using a Tumor Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Single cell suspensions were washed, fixed, stained for the respective immune cell markers, and then analyzed by flow cytometry. Figure 61 shows a comparison of immune cell populations in Y856, LLC-1, and M109 tumors measured according to the above evaluation. (FDH=compound 150, SOC is anti-PD1+chemotherapy (carboplatin+paclitaxel)).

[0457] Treatment with compound 150 significantly increased the population of antitumor macrophages and natural killer (NK) cells, and further reduced the number of tumor-associated macrophages (TAMs) in hot tumor models (M109) but not in cold tumor models (Y8569 and LLC-1). This data supports that compound 150 monotherapy can induce macrophage reprogramming, resulting in a reduction in the number of TAMs and an enhancement of the NK cell population.

Claims

1. formula: TL-L-H ｎ A conjugate having, or a pharmaceutically acceptable salt thereof, During the ceremony, TLs are targeting ligands for target proteins on the surface of viruses, virus-infected cells, cancer cells, immune cells, or fibroblasts. L is a linker, H is a hapten, n is an integer between 2 and 3. At least two of H are optionally capable of binding to different antibodies when in contact with them. Conjugate, or a pharmaceutically acceptable salt thereof.

2. The conjugate according to claim 1, wherein at least two of the H atoms are bound to antibodies.

3. The conjugate according to claim 1, wherein a different antibody is bound to each H.

4. The conjugate according to claim 1, wherein each H is independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof.

5. The conjugate according to claim 1, wherein n is 2.

6. The conjugate according to claim 1, wherein n is 3.

7. The conjugate according to claim 1, wherein each H is independently selected from rhamnose fragment, α-galactosyl moiety, DNP fragment, TNP fragment, fluorescein, digoxigenin, biotin, or from antigens of viruses selected from diphtheria, herpes zoster virus, human papillomavirus, influenza virus, SARS-CoV-2, yellow fever, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis G, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, varicella stomatitis virus, rubella virus, smallpox, monkeypox, poliovirus, dengue virus, and measles virus.

8. The conjugate according to claim 1, wherein n is 2, the first H is a DNP fragment, and the second H is a rhamnose fragment.

9. The conjugate according to claim 1, wherein at least one H is an influenza virus antigen selected from hemagglutinin and neuraminidase.

10. The conjugate according to claim 1, wherein at least one H is a hepatitis antigen selected from L-HBsAg, S-HBsAg, M-HBsAg, and preS.

11. The conjugate according to claim 1, wherein at least one H is gp120 or gp160.

12. The conjugate according to claim 1, wherein at least one H is a glycoprotein.

13. The conjugate according to claim 1, wherein the target protein is a viral envelope protein or a viral envelope protein on the surface of a virus-infected cell.

14. The conjugate according to claim 1, wherein the target protein is influenza neuraminidase or influenza hemagglutinin.

15. The conjugate according to claim 1, wherein the target protein is respiratory syncytium virus fusion protein F.

16. The conjugate according to claim 1, wherein the target protein is a coronavirus spike protein.

17. The conjugate according to claim 1, wherein the target protein is hepatitis B virus surface antigen or HBV core antigen.

18. The conjugate according to claim 1, wherein the target protein is a cell surface receptor on cancer cells.

19. The conjugate according to claim 1, wherein the target protein is a folate receptor.

20. The conjugate according to claim 19, wherein the target protein is folate receptor α or folate receptor β.

21. The conjugate according to claim 1, wherein the target protein is a prostate-specific membrane antigen.

22. The conjugate according to claim 1, wherein the target protein is carbonic anhydrase 9.

23. The conjugate according to claim 1, wherein the target protein is a luteinizing hormone-releasing hormone receptor.

24. The conjugate according to claim 1, wherein the target protein is a neurokinin 1 receptor.

25. The conjugate according to claim 1, wherein the target protein is a cell surface receptor on tumor-associated macrophages.

26. The conjugate according to claim 1, wherein the target protein is a cell surface receptor on myeloid-derived suppressor cells.

27. The conjugate according to claim 1, wherein the target protein is a cell surface receptor on cancer-associated fibroblasts.

28. The conjugate according to claim 1, wherein the target protein is a fibroblast-activating protein.

29. The conjugate according to claim 1, wherein the targeting ligand is a neuraminidase inhibitor.

30. The conjugate according to claim 1, wherein the targeting ligand is an oseltamivir fragment, a zanamivir fragment, a peramivir fragment, or a laninamivir fragment.

31. The conjugate according to claim 1, wherein the targeting ligand is a zanamivir fragment.

32. The conjugate according to claim 1, wherein the targeting ligand is a folic acid fragment or an analogue thereof.

33. The conjugate according to claim 1, wherein the targeting ligand is 5-methyltetrahydrophorate.

34. L is (-CH ２ CH ２ -O-) ｎ The conjugate according to claim 1, comprising a peptide, a peptidoglycan, or a combination of two or more of the above, (wherein n is an integer from 1 to 32, including the values ​​at both ends of the formula).

35. The conjugate according to claim 1, wherein L is a branched linker, and at least two of the haptens are connected to different branches of the linker, the different branches optionally extending from different atoms of the linker.

36. The conjugate according to claim 1, wherein the targeting ligand is a folic acid fragment or a derivative thereof, and at least the first H comprises a rhamnose fragment, and at least the second H comprises a dinitrophenyl fragment.

37. The conjugate according to claim 1, formulated as a prodrug.

38. formula: 【Chemistry 1】 A conjugate having, or a pharmaceutically acceptable salt thereof, During the ceremony, TLs are targeting ligands for target proteins on the surface of viruses, virus-infected cells, cancer cells, immune cells, or fibroblasts. L ａ , L ｂ , and L ｃ These are linkers that may or may not be the same, C is a carbon atom, R ４ is selected from hydrogen, C １ to C ５ alkyl group, C １ to C ５ alkenyl group, or C １ to C ５ alkynyl group, and H １ and H ２ These are haptens, Optionally, H １ and H ２ Each can bind to a different antibody. Conjugate, or a pharmaceutically acceptable salt thereof.

39. formula: 【Chemistry 2】 A conjugate having, and a pharmaceutically acceptable salt thereof, During the ceremony, TLs are targeting ligands for target proteins on the surface of viruses, virus-infected cells, cancer cells, immune cells, or fibroblasts. L ａ , L ｂ , L ｃ , and L ｄ These are linkers that may or may not be the same, C is a carbon atom, H １ , H ２ , and H ３ These are haptens, Optional selection, each H １ H ２ , and H ３ Each can bind to a different antibody. Conjugates and their pharmaceutically acceptable salts.

40. H １ and H ２ The conjugate according to claim 38, wherein an antibody is bound to each of the two parts.

41. H １ H ２ , and H ３ The conjugate according to claim 39, wherein an antibody is bound to each of the two parts.

42. H １ and H ２ The conjugate according to claim 38, wherein each is independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof.

43. H １ H ２ , and H ３ The conjugate according to claim 39, wherein each is independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof.

44. H １ or H ２ The conjugate according to claim 38, wherein each is independently selected from rhamnose fragment, α-galactosyl moiety, DNP fragment, TNP fragment, fluorescein, digoxigenin, biotin, or from antigens of viruses selected from diphtheria, herpes zoster virus, human papillomavirus, influenza virus, SARS-CoV-2, yellow fever, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis G, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, varicella stomatitis virus, rubella virus, smallpox, monkeypox, poliovirus, dengue virus, and measles virus.

45. H １ H ２ , or H ３ The conjugate according to claim 38, wherein each is independently selected from rhamnose fragment, α-galactosyl moiety, DNP fragment, TNP fragment, fluorescein, digoxigenin, biotin, or from antigens of viruses selected from diphtheria, herpes zoster virus, human papillomavirus, influenza virus, SARS-CoV-2, yellow fever, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis G, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, varicella stomatitis virus, rubella virus, smallpox, monkeypox, poliovirus, dengue virus, and measles virus.

46. H １ This is a DNP fragment, H ２ The conjugate according to claim 38, wherein the conjugate is a rhamnose fragment.

47. The conjugate according to claim 38, wherein at least one hapten is an influenza virus antigen selected from hemagglutinin and neuraminidase.

48. The conjugate according to claim 38, wherein at least one hapten is a hepatitis antigen selected from L-HBsAg, S-HBsAg, M-HBsAg, and preS.

49. The conjugate according to claim 38, wherein at least one hapten is gp120 or gp160.

50. The conjugate according to claim 38, wherein at least one hapten is a glycoprotein.

51. The conjugate according to claim 38, wherein the target protein is a viral envelope protein or a viral envelope protein on the surface of a virus-infected cell.

52. The conjugate according to claim 38, wherein the target protein is influenza neuraminidase or influenza hemagglutinin.

53. The conjugate according to claim 38, wherein the target protein is respiratory syncytial virus fusion protein F.

54. The conjugate according to claim 38, wherein the target protein is a coronavirus spike protein.

55. The conjugate according to claim 38, wherein the target protein is hepatitis B virus surface antigen or HBV core antigen.

56. The conjugate according to claim 38, wherein the target protein is a cell surface receptor on cancer cells.

57. The conjugate according to claim 38, wherein the target protein is a folate receptor.

58. The conjugate according to claim 38, wherein the target protein is folate receptor α or folate receptor β.

59. The conjugate according to claim 38, wherein the target protein is a prostate-specific membrane antigen.

60. The conjugate according to claim 38, wherein the target protein is carbonic anhydrase 9.

61. The conjugate according to claim 38, wherein the target protein is a luteinizing hormone-releasing hormone receptor.

62. The conjugate according to claim 38, wherein the target protein is a neurokinin 1 receptor.

63. The conjugate according to claim 38, wherein the target protein is a cell surface receptor on tumor-associated macrophages.

64. The conjugate according to claim 38, wherein the target protein is a cell surface receptor on myeloid-derived suppressor cells.

65. The conjugate according to claim 38, wherein the target protein is a cell surface receptor on cancer-associated fibroblasts.

66. The conjugate according to claim 38, wherein the target protein is a fibroblast-activating protein.

67. The conjugate according to claim 38, wherein the targeting ligand is a neuraminidase inhibitor.

68. The conjugate according to claim 38, wherein the targeting ligand is an oseltamivir fragment, a zanamivir fragment, a peramivir fragment, or a laninamivir fragment.

69. The conjugate according to claim 38, wherein the targeting ligand is a zanamivir fragment.

70. The conjugate according to claim 38, wherein the targeting ligand is a folic acid fragment or an analogue thereof.

71. The conjugate according to claim 38, wherein the targeting ligand is 5-methyltetrahydrophorate.

72. L ａ , L ｂ , L ｃ , and L ｄ At least one of them, independently, (-CH ２ CH ２ -O-) ｎ (In the formula, n is an integer between 1 and 32, including the values ​​at both ends.) alkyl group, peptide, Peptidoglycan, or Two or more combinations of the above The conjugate according to claim 39, including the following:

73. L ａ , L ｂ , and L ｃ At least one of them, independently, (-CH ２ CH ２ -O-) ｎ (In the formula, n is an integer between 1 and 32, including the values ​​at both ends.) alkyl group, peptide, Peptidoglycan, or Two or more combinations of the above The conjugate according to claim 38, including the following:

74. The conjugate according to claim 72, wherein n is an integer from 1 to 16, including the values ​​at both ends.

75. L ａ , L ｂ , and L ｃ , and L ｄ The conjugate according to claim 39, wherein at least one of the members comprises a peptide fragment or a peptidoglycan fragment.

76. L ａ , L ｂ , and L ｃ The conjugate according to claim 39, wherein at least one of the members comprises a peptide fragment or a peptidoglycan fragment.

77. L ａ , L ｂ , and L ｃ , L ｄ Each of them is independent of C ２ ~C １８ The conjugate according to claim 39, comprising an alkyl group.

78. L ａ , L ｂ , and L ｃ Each of them is independent of C ２ ~C １８ The conjugate according to claim 38, comprising an alkyl group.

79. formula: 【Transformation 3】 The conjugate of or a pharmaceutically acceptable salt thereof.

80. formula: 【Chemistry 4】 The conjugate of or a pharmaceutically acceptable salt thereof.

81. The conjugate according to claim 79, wherein one or more of the -OH groups are independently replaced with a thiol, a phosphate, or a phosphanate ester.

82. The conjugate according to claim 79, wherein one or more of the -OH groups are replaced by -OC(=O)R (wherein R is an alkyl group).

83. - One or more of the OH groups are -OC(=O)R (wherein R is C １ ~C ６ The conjugate according to claim 79, wherein it is replaced with an alkyl group.

84. Amine (-NH ２ The group is -OC(=O)R ２ It has been replaced with R ２ The conjugate according to claim 79, wherein is an alkyl group.

85. Amine (-NH ２ The group is -OC(=O)R ２ It has been replaced with R ２ C １ ~C ６ The conjugate according to claim 79, wherein the alkyl group is an alkyl group.

86. The carboxyl (-COOH) group is -OC(=O)R ３ It has been replaced with R ３ The conjugate according to claim 79, wherein is an alkyl group.

87. The carboxyl (-COOH) group is replaced by -OC(=O)R ３ where R ３ is a C １ to C ６ alkyl group, the conjugate according to claim 79.

88. formula: 【Transformation 5】 A conjugate of or a pharmaceutically acceptable salt thereof, wherein L1, L2, and L3 are linkers.

89. L1, L2, and L3, one or more of them, (-CH ２ CH ２ -O-) ｎ The conjugate according to claim 88, comprising (wherein n is an integer from 1 to 16, including the values ​​at both ends).

90. L1, L2, and L3 are each independently C ２ to C １８ The conjugate according to claim 88, comprising an alkyl group, a peptide fragment, or a peptidoglycan fragment.

91. formula: 【Transformation 6】 The conjugate of or a pharmaceutically acceptable salt thereof.

92. The conjugate according to claim 79, which is further conjugated in vivo with one or more antibodies.

93. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 91 and a pharmaceutically acceptable excipient.

94. The pharmaceutical composition according to claim 93 for use in a method of treating a viral infection of a target, comprising administering an effective amount of the pharmaceutical composition to the target.

95. The pharmaceutical composition according to claim 94, further comprising administering an autoantibody or an alloimmunoglobulin G (IgG) antibody to the subject.

96. The pharmaceutical composition according to claim 94, wherein the viral infection is influenza.

97. The pharmaceutical composition according to claim 94, wherein the pharmaceutical composition is administered orally.

98. The pharmaceutical composition according to claim 94, wherein the pharmaceutical composition is administered once a day.

99. The pharmaceutical composition according to claim 94, wherein the pharmaceutical composition is administered multiple times a day.

100. The pharmaceutical composition according to claim 94, wherein the pharmaceutical composition is administered twice a day.

101. The pharmaceutical composition according to claim 93 for use in a method of treating a target cancer, comprising administering an effective amount of the pharmaceutical composition to the target.

102. The pharmaceutical composition according to claim 101, wherein the method further comprises administering an autoantibody or an alloglobulin antibody to the subject.

103. The pharmaceutical composition according to claim 101, wherein the cancer is a hot cancer.

104. The pharmaceutical composition according to claim 101, wherein the cancer is kidney cancer, lung cancer, or colorectal cancer.

105. The pharmaceutical composition according to claim 94, wherein the pharmaceutical composition is administered orally or intravenously.

106. The pharmaceutical composition according to claim 94, wherein the pharmaceutical composition is administered once a day.

107. The pharmaceutical composition according to claim 94, wherein the method further comprises administering a second therapeutic agent to the subject, the second therapeutic agent comprising a chemotherapeutic agent, sunitinib, a PD-1 inhibitor, or a PDL-1 inhibitor.

108. The pharmaceutical composition according to claim 93, for use in a method for activating an immune response in a target, which includes administering an effective amount of the pharmaceutical composition to the target.

109. The pharmaceutical composition according to claim 108, wherein the immune response is an innate immune response.

110. The pharmaceutical composition according to claim 108, wherein the immune response is activated within the target region of the subject, and the target region is the tumor microenvironment or the location of a viral replication site.

111. The pharmaceutical composition according to claim 108, wherein the method further comprises administering an autoantibody or an alloglobulin antibody to the subject.

112. The pharmaceutical composition according to claim 110, wherein administration of an effective amount of the conjugate or the pharmaceutical composition induces reprogramming of M2 type macrophages into M1 type macrophages in the target region.

113. The conjugate according to claim 1, wherein at least two of the H components can each bind to different antibodies when in vivo contact with an antibody.

114. The conjugate according to claim 1, wherein two of the H atoms can each bind to different antibodies when in vitro contact with an antibody.

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